Germline gain-of-function mutations in SOS1 cause Noonan syndrome.

Noonan syndrome, the most common single-gene cause of congenital heart disease, is characterized by short stature, characteristic facies, learning problems and leukemia predisposition. Gain-of-function mutations in PTPN11, encoding the tyrosine phosphatase SHP2, cause approximately 50% of Noonan syndrome cases. SHP2 is required for RAS-ERK MAP kinase (MAPK) cascade activation, and Noonan syndrome mutants enhance ERK activation ex vivo and in mice. KRAS mutations account for <5% of cases of Noonan syndrome, but the gene(s) responsible for the remainder are unknown. We identified missense mutations in SOS1, which encodes an essential RAS guanine nucleotide-exchange factor (RAS-GEF), in approximately 20% of cases of Noonan syndrome without PTPN11 mutation. The prevalence of specific cardiac defects differs in SOS1 mutation-associated Noonan syndrome. Noonan syndrome-associated SOS1 mutations are hypermorphs encoding products that enhance RAS and ERK activation. Our results identify SOS1 mutants as a major cause of Noonan syndrome, representing the first example of activating GEF mutations associated with human disease and providing new insights into RAS-GEF regulation.

Impaired lymphatic contraction associated with immunosuppression.

To trigger an effective immune response, antigen and antigen-presenting cells travel to the lymph nodes via collecting lymphatic vessels. However, our understanding of the regulation of collecting lymphatic vessel function and lymph transport is limited. To dissect the molecular control of lymphatic function, we developed a unique mouse model that allows intravital imaging of autonomous lymphatic vessel contraction. Using this method, we demonstrated that endothelial nitric oxide synthase (eNOS) in lymphatic endothelial cells is required for robust lymphatic contractions under physiological conditions. By contrast, under inflammatory conditions, inducible NOS (iNOS)-expressing CD11b(+)Gr-1(+) cells attenuate lymphatic contraction. This inhibition of lymphatic contraction was associated with a reduction in the response to antigen in a model of immune-induced multiple sclerosis. These results suggest the suppression of lymphatic function by the CD11b(+)Gr-1(+) cells as a potential mechanism of self-protection from autoreactive responses during on-going inflammation. The central role for nitric oxide also suggests that other diseases such as cancer and infection may also mediate lymphatic contraction and thus immune response. Our unique method allows the study of lymphatic function and its molecular regulation during inflammation, lymphedema, and lymphatic metastasis.

Medical complications, clinical findings, and educational outcomes in adults with Noonan syndrome.

Noonan syndrome (NS) is a heterogeneous developmental disorder caused by missense mutations in genes involved in the Ras/MAPK signaling pathway, a major mediator of early and late developmental processes. The diagnosis of NS is made on clinical grounds with molecular confirmation of a mutation found in 63% of cases. Key clinical features include short stature, cardiac defects, developmental delay, lymphatic dysplasias, bleeding tendency, and a constellation of distinctive facial features and physical exam findings. The prevalence of medical issues or the development of new ones in adults with NS is not well-studied. This cross-sectional study reports on the prevalence of clinical conditions and their ages of onset in a cohort of 35 adolescents and adults with NS aged 16-68 years old (mean age 28 years). In this cohort, 34 of 35 subjects (97%) had had full PTPN11 sequencing; 37% were PTPN11 positive, 23% were SOS1 positive, and 3% were BRAF positive. Mean adult height in both men and women was at the 3rd-10th centile. The most prevalent clinical findings in this cohort included pulmonary valve stenosis (71%), easy bruising (63%), GERD (60%), constipation (51%), scoliosis (54%), chronic joint pain (54%), lymphedema (49%), depression (49%), anxiety (49%), Chiari malformation (20%), and osteopenia/osteoporosis (14%). In summary, adults with NS are affected by multi-organ morbidity and require special medical management aimed towards the most prevalent and serious known medical complications. Larger studies characterizing the clinical conditions found in NS adults are needed to provide potential genotype-phenotype correlations that may aid in clinical management.

Optical imaging with a novel cathepsin-activatable probe for enhanced detection of colorectal cancer.

We evaluated a cysteine cathepsin-activatable optical imaging probe (LUM015) with improved kinetics relative to larger macromolecules for detection and characterization of colorectal cancer (CRC), and thereby assessed its potential use in fluorescence-guided colonoscopy. We showed that LUM015 is stable in plasma. In-vitro studies demonstrated selectivity of LUM015 for targeting cathepsins; there was robust increase in emitted fluorescence signal from the cathepsin overexpressing HT-29 CRC cells within 1-5 minutes after incubation with LUM015 compared to the cells incubated with combination of LUM015 and a pan-protease inhibitor (as negative control). Biodistribution, differential accumulation of the probe in the tumor and tumor-to-background fluorescence signal ratio of LUM015 were compared to ProSense680, a commercially available protease-activatable optical imaging probe, over 24 hours after intravenous injection of the probes in nude mice with subcutaneously implanted HT-29 tumors. LUM015 showed distinct kinetics compared to ProSense680 with time to peak signal for subcutaneous tumor-to-colon ratio of 3.3±0.3 (mean ± SD) at 4-8 hours compared to 2.9±0.2 at 24 hours, respectively (n=8 for each group). Near-infrared fluorescence imaging and dual channel colonoscopy of the mice with orthotopic colon tumors showed tumor-to-colon ratio of 3.7±0.2 in HT-29 tumors (n=4), 2.8±0.1 in genetically engineered mice with APCKOKrasLSL-G12Dp53flox/flox mutation (n=4), and 4.1±0.1 in mice with APCLoxP/LoxPMsh2LoxP/LoxP mutation (n=4) at 6 hours after LUM015 administration. Immunohistochemistry and laser confocal microscopy of the extracted tumors confirmed high expression of cysteine cathepsins in all colon tumor types tested. Optical imaging with cathepsin-activatable LUM015 in multiple models of CRC highlights its potential for increasing the efficacy of CRC screening and therapeutic procedures.

Inactivation of conditional Rb by Villin-Cre leads to aggressive tumors outside the gastrointestinal tract.

We have crossed mice carrying the conditional Rb(tm2Brn) allele with a constitutive Villin-Cre transgenic mouse. The Villin promoter in these animals is highly expressed in adult intestine and kidney proximal tubules and is expressed in the gut and nephros anlagen during embryogenesis. We report here that these mice develop tumors between 12 and 17 months old outside the gastrointestinal (GI) tract. A high penetrance of pituitary tumors and medullar carcinoma of the thyroid is observed with a lower incidence of hyperplasia of pulmonary neuroendocrine cells and aggressive liver, bile duct, stomach, oral cavity tumors, and lipomas. Rb rearrangement due to ectopic Villin promoter activity in neural crest or neural crest stem cells during embryogenesis is most likely responsible for the medullar carcinoma of the thyroid phenotype. The aggressive nature of the medullar carcinoma of the thyroid and its ability to metastasize to unusual sites make the model suitable for the study of tumor progression and mechanism of metastasis. Observed sites of metastasis include the stomach, small intestine, liver, lung, kidney, pancreas, spleen, bone marrow, salivary gland, fat, lymph nodes, and dorsal root ganglion. Because the Villin promoter is highly active throughout the GI and in the nephros anlagen during development, we find that Rb inactivation is not sufficient to initiate tumorigenesis in the GI or kidneys in mice.

Genomic and expression analysis of the 12p11-p12 amplicon using EST arrays identifies two novel amplified and overexpressed genes.

We performed parallel array comparative genomic hybridization and array expression analysis of the 12p11-p12 amplicon in human testicular seminomas and an ovarian carcinoma cell line using an expressed sequence tags (ESTs) array spotted with 8254 ESTs. The data were normalized using a robust statistical modeling and the significance inferred from the local SD. We identified two ESTs within the chromosomal amplicon that were amplified and overexpressed in > or =75-100 percent of analyzed tumors with the 12p11-p12 amplicon. These sequences, belonging to coding regions of two novel genes designated here as GCT1 and GCT2, were broadly expressed in a panel of human tissues, including testis and ovary. GCT1 and GCT2 were overexpressed in 92 and 71 percent, respectively, of a panel of seminomas tested. Combined array comparative genomic hybridization and array expression analysis is a valid approach for gene discovery in large chromosomal amplicons.

Cardiovascular disease in Noonan syndrome.

BACKGROUND: Noonan syndrome (NS), a relatively common autosomal dominant disorder with an incidence of 1 in 1000 to 2500 live births, is the most common syndromic cause of congenital heart disease after Trisomy 21. OBJECTIVE: To comprehensively define the spectrum of cardiac morphology and specific clinical course of a large cohort of NS patients. DESIGN: Retrospective, descriptive case series study. PATIENTS: An international Harvard-based NS registry was combined with clinical data from NS patients followed at Boston Children's Hospital, Massachusetts, USA. RESULTS: We identified 293 patients with NS. Cardiovascular disease was seen in 81% (n=237) including pulmonary stenosis in 57%, secundum atrial septal defects in 32% and hypertrophic cardiomyopathy in 16%. A genetic mutation of the RAS-MAPK signalling pathway was identified in 62% (n=136). Genotype-phenotype associations were noted between PTPN11 mutations and atrial septal defects (p=0.001), and pulmonary stenosis (p<0.001). RAF1 mutations were associated with hypertrophic cardiomyopathy (p<0.001). Cardiovascular outcomes that differed specifically in a NS cohort included high re-intervention rates (65%) after percutaneous balloon pulmonary valvuloplasty for valvar pulmonary stenosis. Additionally, in NS patients with hypertrophic cardiomyopathy, a clinically significant regression of hypertrophy (17%) was observed as was a markedly higher incidence of concomitant congenital heart defects (70%). CONCLUSIONS: Patients with NS have a distinct spectrum of cardiac phenotypes that may have a natural history and response to therapy atypical to that normally seen in non-syndromic heart disease. A diagnosis of NS in a patient with pulmonary stenosis or infant-onset hypertrophic cardiomyopathy would facilitate condition-specific counselling on outcome and prognosis.

Genotype directed therapy in murine mismatch repair deficient tumors.

The PI3K/AKT/mTOR pathway has frequently been found activated in human tumors. We show that in addition to Wnt signaling dysfunction, the PI3K/AKT/mTOR pathway is often upregulated in mouse Msh2(-/-) initiated intestinal tumors. NVP-BEZ235 is a dual PI3K/mTOR inhibitor toxic to many cancer cell lines and currently involved in clinical trials. We have treated two mouse models involving Msh2 that develop small intestinal and/or colonic tumors with NVP-BEZ235, and a subset of animals with NVP-BEZ235 and MEK inhibitor ADZ4266. The disease phenotype has been followed with pathology, (18)F FDG PET imaging, and endoscopy. Intestinal adenocarcinomas are significantly decreased in multiplicity by both drug regimens. The majority of tumors treated with combined therapy regress significantly, while a small number of highly progressed tumors persist. We have examined PTEN, AKT, MEK 1&2, MAPK, S6K, mTOR, PDPK1, and Cyclin D1 and find variable alterations that include downregulation of PTEN, upregulation of AKT and changes in its phosphorylated forms, upregulation of pMEK 1&2, p42p44MAPK, pS6K, and Cyclin D1. Apoptosis has been found intact in some tumors and not in others. Our data indicate that NVP-BEZ235 alone and in combination with ADZ4266 are effective in treating a proportion of colorectal cancers, but that highly progressed resistant tumors grow in the presence of the drugs. Pathways upregulated in some resistant tumors also include PDPK1, suggesting that metabolic inhibitors may also be useful in treating these tumors.

A restricted spectrum of NRAS mutations causes Noonan syndrome.

Noonan syndrome, a developmental disorder characterized by congenital heart defects, reduced growth, facial dysmorphism and variable cognitive deficits, is caused by constitutional dysregulation of the RAS-MAPK signaling pathway. Here we report that germline NRAS mutations conferring enhanced stimulus-dependent MAPK activation account for some cases of this disorder. These findings provide evidence for an obligate dependency on proper NRAS function in human development and growth.

PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity.

Noonan syndrome (NS) is a developmental disorder characterized by facial dysmorphia, short stature, cardiac defects, and skeletal malformations. We recently demonstrated that mutations in PTPN11, the gene encoding the non-receptor-type protein tyrosine phosphatase SHP-2 (src homology region 2-domain phosphatase-2), cause NS, accounting for approximately 50% of cases of this genetically heterogeneous disorder in a small cohort. All mutations were missense changes and clustered at the interacting portions of the amino-terminal src-homology 2 (N-SH2) and protein tyrosine phosphatase (PTP) domains. A gain of function was postulated as a mechanism for the disease. Here, we report the spectrum and distribution of PTPN11 mutations in a large, well-characterized cohort with NS. Mutations were found in 54 of 119 (45%) unrelated individuals with sporadic or familial NS. There was a significantly higher prevalence of mutations among familial cases than among sporadic ones. All defects were missense, and several were recurrent. The vast majority of mutations altered amino acid residues located in or around the interacting surfaces of the N-SH2 and PTP domains, but defects also affected residues in the C-SH2 domain, as well as in the peptide linking the N-SH2 and C-SH2 domains. Genotype-phenotype analysis revealed that pulmonic stenosis was more prevalent among the group of subjects with NS who had PTPN11 mutations than it was in the group without them (70.6% vs. 46.2%; P<.01), whereas hypertrophic cardiomyopathy was less prevalent among those with PTPN11 mutations (5.9% vs. 26.2%; P<.005). The prevalence of other congenital heart malformations, short stature, pectus deformity, cryptorchidism, and developmental delay did not differ between the two groups. A PTPN11 mutation was identified in a family inheriting Noonan-like/multiple giant-cell lesion syndrome, extending the phenotypic range of disease associated with this gene.