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Germline mutations in PTPN11, the gene encoding the Src-homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu

GJA5 gene (MIM no. 121013), localized at 1q21.1, encodes for the cardiac gap junction protein connexin 40. In humans, copy number variants of chromosome 1q21.1 have been associated with variable phenotypes comprising congenital heart disease (CHD), including isolated TOF. In mice, the deletion of Gja5 can cause a variety of complex CHDs, in particular of the cardiac outflow tract, corresponding to TOF in many cases. In the present study, we screened for mutations in the GJA5 gene 178 unrelated probands with isolated TOF. A heterozygous nucleotide change (c.793C>T) in exon 2 of the gene leading to the p.Pro265Ser variant at the carboxyl-terminus of the protein was found in two unrelated sporadic patients, one with classic anatomy and one with pulmonary atresia. This GJA5 missense substitution was not observed in 1568 ethnically-matched control chromosomes. Immunofluorescent staining and confocal microscopy revealed that cells expressing the mutant protein form sparse or no visible gap-junction plaques in the region of cell-cell contact. Moreover, analysis of the transfer of the gap junction permanent tracer lucifer yellow showed that cells expressing the mutant protein have a reduced rate of dye transfer compared with wild-type cells. Finally, use of a zebrafish model revealed that microinjection of the GJA5-p.Pro265Ser mutant disrupts overall morphology of the heart tube in the 37% (22/60) of embryos, compared with the 6% (4/66) of the GJA5 wild-type-injected embryos. These findings implicate GJA5 gene as a novel susceptibility gene for TOF.

Telethon-Italy ERA-Net for research programs on rare diseases 2009 (European network on Noonan Syndrome and related disorders) Associazione Italiana Sindromi di Costello e Cardiofaciocutanea NIH Italian Ministry of Health Italian Ministry of Education, University and Research Noonan syndrome (NS) is among the most common nonchromosomal disorders affecting development and growth. NS is caused by aberrant RAS-MAPK signaling and is genetically heterogeneous, which explains, in part, the marked clinical variability documented for this Mendelian trait. Recently, we and others identified SOS1 as a major gene underlying NS. Here, we explored further the spectrum of SOS1 mutations and their associated phenotypic features. Mutation scanning of the entire SOS1 coding sequence allowed the identification of 33 different variants deemed to be of pathological significance, including 16 novel missense changes and in-frame indels. Various mutation clusters destabilizing or altering orientation of regions of the protein predicted to contribute structurally to the maintenance of autoinhibition were identified. Two previously unappreciated clusters predicted to enhance SOS1's recruitment to the plasma membrane, thus promoting a spatial reorientation of domains contributing to inhibition, were also recognized. Genotype-phenotype analysis confirmed our previous observations, establishing a high frequency of ectodermal anomalies and a low prevalence of cognitive impairment and reduced growth. Finally, mutation analysis performed on cohorts of individuals with nonsyndromic pulmonic stenosis, atrial septal defects, and ventricular septal defects excluded a major contribution of germline SOS1 lesions to the isolated occurrence of these cardiac anomalies. Hum Mutat 32:760-772, 2011. (C) 2011 Wiley-Liss, Inc. Ist Super Sanita, Dept Hematol Oncol & Mol Med, I-00161 Rome, Italy IRCCS Casa Sollievo Sofferenza, Lab Mendel, San Giovanni Rotondo, Italy Univ Roma Tor Vergata, Dipartimento Sci & Tecnol Chim, Rome, Italy St Orsola Marcello Malpighi Hosp, UO Genet Med, Bologna, Italy Univ Turin, Dipartimento Pediat, Turin, Italy GeneDx, Gaithersburg, MD USA Universidade Federal de São Paulo, São Paulo, Brazil Univ Bari, Dept Biomed Dev Age, Bari, Italy Osped Pediat, Brescia, Italy Osped Bolzano, Serv Aziendale Consulenza Genet, Bolzano, Italy Ospedali Galliera, SC Genet Umana, Genoa, Italy Univ Politecn Marche, Ist Sci Materno Infantili, Ancona, Italy Univ Roma La Sapienza, Dept Pediat, Div Pediat Cardiol, Rome, Italy Univ Cattolica Sacro Cuore, Ist Genet Med, Rome, Italy Univ Milano Bicocca, AOS Gerardo Fdn MBBM, Pediat Clin, Monza, Italy Univ Bologna, Dipartimento Pediat, Bologna, Italy IRCCS, Osped Pediat Bambino Gesu, Rome, Italy Univ Cattolica Sacro Cuore, Ist Clin Pediat, Rome, Italy Mt Sinai Sch Med, Child Hlth & Dev Inst, New York, NY USA Universidade Federal de São Paulo, EPM, São Paulo, Brazil Telethon-Italy: GGP07115 Telethon-Italy: GGP10020 NIH: HL71207 Italian Ministry of Health: RC2009 Italian Ministry of Health: RC2010 Italian Ministry of Education, University and Research: FIRB RBIP06PMF2_005 Web of Science

N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue(1,2). We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat-containing protein that positively modulates RAS-MAPK signal flow(3-6), underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721)(7) shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2(S2G) in vitro enhanced MAPK activation in a cell typespecific fashion. Induction of SHOC2(S2G) in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.

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 approximately 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.