Giorgia Esposito

Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes ∼60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non–HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

Germline BRAF mutations in noonan, LEOPARD, and cardiofaciocutaneous Syndromes: Molecular diversity and associated phenotypic spectrum

Noonan, LEOPARD, and cardiofaciocutaneous syndromes (NS, LS, and CFCS) are developmental disorders with overlapping features including distinctive facial dysmorphia, reduced growth, cardiac defects, skeletal and ectodermal anomalies, and variable cognitive deficits. Dysregulated RAS–mitogen‐activated protein kinase (MAPK) signal traffic has been established to represent the molecular pathogenic cause underlying these conditions. To investigate the phenotypic spectrum and molecular diversity of germline mutations affecting BRAF, which encodes a serine/threonine kinase functioning as a RAS effector frequently mutated in CFCS, subjects with a diagnosis of NS (N=270), LS (N=6), and CFCS (N=33), and no mutation in PTPN11, SOS1, KRAS, RAF1, MEK1, or MEK2, were screened for the entire coding sequence of the gene. Besides the expected high prevalence of mutations observed among CFCS patients (52%), a de novo heterozygous missense change was identified in one subject with LS (17%) and five individuals with NS (1.9%). Mutations mapped to multiple protein domains and largely did not overlap with cancer‐associated defects. NS‐causing mutations had not been documented in CFCS, suggesting that the phenotypes arising from germline BRAF defects might be allele specific. Selected mutant BRAF proteins promoted variable gain of function of the kinase, but appeared less activating compared to the recurrent cancer‐associated p.Val600Glu mutant. Our findings provide evidence for a wide phenotypic diversity associated with mutations affecting BRAF, and occurrence of a clinical continuum associated with these molecular lesions. Hum Mutat 0:1–8, 2009.

Spectrum of atrial septal defects associated with mutations of NKX2.5 and GATA4 transcription factors

Atrial septal defect (ASD) is a common cardiovascular malformation, affecting over 1 in 1000 live births, accounting for 10% of congenital heart defects (CHD).1 ASD refers to a communication between the right and left atria, anatomically classified into the deficient atrial septum structure. ASD ostium secundum (ASDos) is the prevalent defect, representing 85% of all ASDs.2 ASD may be isolated or associated with other CHDs, such as pulmonary valve stenosis (PVS), ventricular septal defect (VSD), or conduction defects. In addition, persistent left to right blood shunt may result in atrial and ventricular dysfunctions and atrial arrhythmias, in the absence of surgical or catheter based repair. The atrial septum is one of the cardiac structures most sensitive to environmental or genetic factors. Several lines of evidence have highlighted a role for different proteins and transcription factors in the septogenesis process;3 however, only two genes, encoding for the transcription factors NKX2.5 and GATA4, have been implicated so far in non-syndromic ASDs.4,5 Clinical and molecular analyses have shown that mutations in these two genes are responsible for ASDs in association with distinct cardiac features.4–15 Mutations in NKX2.5 , a member of the NK-2 class of homeobox genes, have been described in autosomal dominant ASDos with progressive atrioventricular (AV) block, and in 1–4% of sporadic ASD patients.4,6–12 Most of these mutations occur within the homeodomain, a critical protein domain that interacts specifically with DNA, and are associated with conduction anomalies. Low penetrance NKX2.5 gene mutations, mainly outside the homeodomain, have been found in 5% of patients with tetralogy of Fallot, and in a number of individuals with other CHDs and normal conduction.4,6,9–13 Recently, heterozygous mutations in the GATA4 zinc finger transcription factor gene have been identified in three families with …

GATA4 mutations in 357 unrelated patients with congenital heart malformation.

Congenital heart disease (CHD) represents one of the most common birth defects, but the genetic causes remain largely unknown. Mutations in GATA4, encoding a zinc finger transcription factor with a pivotal role in heart development, have been associated with CHD in several familial cases and a small subset of sporadic patients. To estimate the pathogenetic role of GATA4 in CHD, we screened for mutations in 357 unrelated patients with different congenital heart malformations. In addition to nine synonymous changes, we identified two known (A411V and D425N) and two novel putative mutations (G69D and P163R) in five patients with atrial or ventricular septal defects that were not seen in control subjects. The four mutations did not show altered GATA4 transcriptional activity in synergy with the transcription factors NKX2-5 and TBX20. Our data expand the spectrum of mutations associated with cardiac septal defects but do not support GATA4 mutations as a common cause of CHD.

A novel PTPN11 gene mutation bridges Noonan syndrome, multiple lentigines/LEOPARD syndrome and Noonan-like/multiple giant cell lesion syndrome

Noonan (NS) and multiple lentigines/LEOPARD syndromes (LS) have proved to be associated with distinct PTPN11 mutations. Noonan-like/multiple giant cell lesion syndrome (NLS) is a rare disease, characterised by short stature, facial dysmorphisms, congenital heart defect (CHD) and central giant cell lesions. PTPN11 gene mutations have been reported in a single NLS family and two sporadic patients. Here we report a patient with a complex phenotype progressing throughout the years from NS at birth towards LS and NLS. PTPN11 gene analysis disclosed a novel missense mutation (Ala461Thr) in exon 12, affecting the consensus sequence of the SHP2-active site. This observation joins together NS and LS to NLS into a unique genetic defect, broadening the clinical and molecular spectrum of PTPN11-related disorders.

Costello syndrome: clinical diagnosis in the first year of life

We report on three patients with Costello syndrome (CS) diagnosed during the first year of life and try to outline the clinical characteristics facilitating early recognition of this syndrome, which can now be corroborated by testing the HRAS gene. Phenotypical overlap of CS with Noonan (NS) and cardiofaciocutaneous syndrome (CFCS), particularly in neonatal age, is well known. Diagnostic features useful for recognition of CS in the first year of life are the following: (1) fetal and neonatal macrosomia with subsequent slow growth due to severe feeding difficulties, (2) developmental delay, (3) particularly coarse facial dysmorphisms and gingival hyperplasia, (4) skeletal anomalies as osteoporosis and metaphyseal enlargement, (5) hypertrophic cardiomyopathy (HCM) with asymmetric septal thickening and systolic anterior motion of the mitral valve, and (6) specific atrial arrhythmias. Following a clinical suspect of CS based on specific features, molecular screening of HRAS gene mutations should precede analysis of the other genes in the Ras-MAPK pathway implicated in related disorders with overlapping phenotypes.

CRELD1 and GATA4 gene analysis in patients with nonsyndromic atrioventricular canal defects.

Anna Sarkozy,* Giorgia Esposito, Emanuela Conti, Maria Cristina Digilio, Bruno Marino, Raffaele Calabrò, Antonio Pizzuti, and Bruno Dallapiccola CSS Hospital, IRCCS, San Giovanni Rotondo, Italy, and CSSMendel Institute, Rome, Italy Department of Experimental Medicine and Pathology, University ‘‘La Sapienza,’’ Rome, Italy Division of Medical Genetics, Bambino Gesù Hospital, IRCCS, Rome, Italy Section of Pediatric Cardiology, Department of Pediatrics, University ‘‘La Sapienza,’’ Rome, Italy Department of Cardiology, Second University of Naples, A.O. Monaldi, Naples, Italy

Somatic mutations in NKX2–5, GATA4, and HAND1 are not a common cause of tetralogy of Fallot or hypoplastic left heart

The majority of congenital heart disease (CHD) occurs as a sporadic finding, with a minority of cases associated with a known genetic abnormality. Combinations of genetic and environmental factors are implicated, with the recent and intriguing hypothesis that an apparently high rate of somatic mutations might explain some sporadic CHD. We used samples of right ventricular myocardium from patients undergoing surgical repair of tetralogy of Fallot (TOF) and hypoplastic left heart (HLH) to examine the incidence of somatic mutation in cardiac tissue. TOF is a common form of cyanotic CHD, occurring in 3.3 per 10,000 live births. HLH is a rare defect in which the left side of the heart is severely under‐developed. Both are severe malformations whose genetic etiology is largely unknown. We carried out direct sequence analysis of the NKX2–5 and GATA4 genes from fresh frozen cardiac tissues and matched blood samples of nine TOF patients. Analysis of NKX2–5, GATA4, and HAND1 was performed from cardiac tissue of 24 HLH patients and three matched blood samples. No somatic or germline mutations were identified in the TOF or HLH patients. Although limited by sample size, our study suggests that somatic mutations in NKX2–5 and GATA4 are not a common cause of isolated TOF or HLH.

Nonsyndromic pulmonary valve stenosis and the PTPN11 gene.

Isolated pulmonary valve stenosis (PVS) is a congenital heart defect (CHD) with an estimated frequency of 4/10,000 live births, and an incidence of about 10% of all children with CHD [Pierpont and Moller, 1987]. Typical PVS consists of a domed form fusion of pulmonary semilunar valves, with poststenotic dilatation of the main pulmonary artery, which is a common form of right ventricular outflow tract obstruction [Ferencz et al., 1997]. PVS can be associated with major cardiovascular defects, but frequently occurs as an isolated malformation, with uniform anatomic characteristics. About 10% of patients with PVS display additional cardiac anomalies [Ferencz et al., 1997], mainly in the formofmalformationsyndromes, including fetalrubella, fetal cytomegalovirus infection (CMV), Ehlers-Danlos, Goldenhar, Costello, and others [Burn and Goodship, 2002]. However, PVS is the classic CHD associated with Noonan (NS) and multiple lentigines/LEOPARD (ML/LEOPARD) syndromes. In these patients, the valvular stenosis usually consists of a characteristic valve leaflet dysplasia [Ferencz et al., 1997]. The dysplastic form of PVS is less common than the typical valve stenosis, and is more commonly associated with noncardiac malformations (such as NS), which are unusual in isolated PVS. Moreover, recurrence of nonsyndromic PVS has been reported in several families, and both syndromic and nonsyndromic familial valve stenosis are inherited as autosomal dominant traits [Pierpont and Moller, 1987]. The risk of recurrence in sporadic cases of nonsyndromic PVS is 2% [Burn and Goodship, 2002]. Recently, NS and ML/LEOPARD syndromes have been related toPTPN11 genemutations [Tartaglia et al., 2001; Digilio et al., 2002], in accordance with animal models, which have shown that PTPN11 gene mutations are associated with dysplastic PVS with thickened semilunar valves, comparable to what observed in NS patients [Chen et al., 2000]. PTPN11 mutations have been identified in 37% of sporadic and 59% of familial NS patients, and in nine of 10 ML/ LEOPARD individuals [Digilio et al., 2002; Tartaglia et al., 2002]. The genotype–phenotype correlation study hasdemonstrateda significant associationbetweenPVS and PTPN11 mutations [Tartaglia et al., 2002]. In order to investigate the precise role of PTPN11 mutations in the pathogenesis of the nonsyndromic, nondysplastic form of valve stenosis, we gathered patients with nonsyndromic PVS and investigated for PTPN11 gene mutations. Twenty affected patients were selected. Informed consent and family history were obtained from all subjects and their parents. All patients were evaluated to exclude syndromic conditions, in particular Noonan and ML/LEOPARD syndromes. Sixteen patients were sporadic, and four were familial. The cardiac diagnosis wasmade by echocardiography and/or cardiac catheterization, or surgery. In all patients, the right ventricular-to-pulmonary artery peak-to-peak gradient exceeded 40 mmHg at Doppler echocardiography. No cases showed dysplasia of the pulmonary valve leaflets. Patients included nine females (45%) and 11males (55%). Ages ranged between 0.2 and 34 years (mean age SD1⁄4 8.4 4.6 years). PTPN11coding regions with exon-intron boundaries (GenBankAccessionNumber: NM_008234)were amplified from genomic DNA by polymerase chain reaction (PCR) and analyzed by single strand conformation polymorfism (SSCP) (Genephor Unit; Amersham-Pharmacia Biotech, Uppsala, Sweden). Fragments with anomalous mobility shifts were sequenced (ABI PRISM 310 Genetic Analyzer automated sequencer; Applied Biosystems, Foster City, CA). No fragment with an aberrant migration pattern was identified by these methods. This result suggests that PTPN11mutations occur only in patients with PVS and NS or ML/LEOPARD syndromes, and not in patients with nonsyndromic defects. Even considering that neither the noncoding regions of the gene nor wide deletions and rearrangements were investigated, these results are interesting. In fact, all PTPN11 mutations reported to date in syndromic patients with or without PVS were of the missense type, with a detection rate ranging from 45% using denaturing high-performance liquid chromatography (dHPLC), to slightly over 50% using direct sequencing [Tartaglia et al., 2001, 2002]. Grant sponsor: Italian Ministry of Health; Grant sponsor: Italian Ministry of Instruction, University, and Research.

Molecular analysis of PRKAG2, LAMP2, and NKX2‐5 genes in a cohort of 125 patients with accessory atrioventricular connection

Molecular Analysis of PRKAG2, LAMP2, and NKX2-5 Genes in a Cohort of 125 Patients With Accessory Atrioventricular Connection Giorgia Esposito, Giorgia Grutter, Fabrizio Drago, Mauro W. Costa, Antonella De Santis, Giovanna Bosco, Bruno Marino, Emanuele Bellacchio, Francesca Lepri, Richard P. Harvey, Anna Sarkozy,* and Bruno Dallapiccola IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, Rome, Italy Casa Sollievo della Sofferenza-Mendel Institute, Rome, Italy Department of Experimental Medicine and Pathology, University ‘‘La Sapienza’’, Rome, Italy Department of Pediatric Cardiology, Bambino Gesu? Hospital, IRCCS, Rome, Italy Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, Australia Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Section of Pediatric Cardiology, Department of Pediatrics, University ‘‘La Sapienza’’, Rome, Italy