Ras/MAPK dysregulation in development causes a skeletal myopathy in an activating BrafL597V mouse model for cardio-facio-cutaneous syndrome.

BACKGROUND: Cardio-facio-cutaneous (CFC) syndrome is a human multiple congenital anomaly syndrome that is caused by activating heterozygous mutations in either BRAF, MEK1, or MEK2, three protein kinases of the Ras/mitogen-activated protein kinase (MAPK) pathway. CFC belongs to a group of syndromes known as RASopathies. Skeletal muscle hypotonia is a ubiquitous phenotype of RASopathies, especially in CFC syndrome. To better understand the underlying mechanisms for the skeletal myopathy in CFC, a mouse model with an activating BrafL597V allele was utilized. RESULTS: The activating BrafL597V allele resulted in phenotypic alterations in skeletal muscle characterized by a reduction in fiber size which leads to a reduction in muscle size which are functionally weaker. MAPK pathway activation caused inhibition of myofiber differentiation during embryonic myogenesis and global transcriptional dysregulation of developmental pathways. Inhibition in differentiation can be rescued by MEK inhibition. CONCLUSIONS: A skeletal myopathy was identified in the CFC BrafL597V mouse validating the use of models to study the effect of Ras/MAPK dysregulation on skeletal myogenesis. RASopathies present a novel opportunity to identify new paradigms of myogenesis and further our understanding of Ras in development. Rescue of the phenotype by inhibitors may help advance the development of therapeutic options for RASopathy patients.

Deficiency of acyl-CoA synthetase 5 is associated with a severe and treatable failure to thrive of neonatal onset.

Failure to thrive (FTT) causes significant morbidity, often without clear etiologies. Six individuals of a large consanguineous family presented in the neonatal period with recurrent vomiting and diarrhea, leading to severe FTT. Standard diagnostic work up did not ascertain an etiology. Autozygosity mapping and whole exome sequencing identified homozygosity for a novel genetic variant of the long chain fatty acyl-CoA synthetase 5 (ACSL5) shared among the affected individuals (NM_203379.1:c.1358C>A:p.(Thr453Lys)). Autosomal recessive genotype-phenotype segregation was confirmed by Sanger sequencing. Functional in vitro analysis of the ACSL5 variant by immunofluorescence, western blotting and enzyme assay suggested that Thr453Lys is a loss-of-function mutation without any remaining activity. ACSL5 belongs to an essential enzyme family required for lipid metabolism and is known to contribute the major activity in the mouse intestine. Based on the function of ACSL5 in intestinal long chain fatty acid metabolism and the gastroenterological symptoms, affected individuals were treated with total parenteral nutrition or medium-chain triglyceride-based formula restricted in long-chain triglycerides. The patients responded well and follow up suggests that treatment is only required during early life.

Senescence in RASopathies, a possible novel contributor to a complex pathophenoype.

Senescence is a biological process that induces a permanent cell cycle arrest and a specific gene expression program in response to various stressors. Following studies over the last few decades, the concept of senescence has evolved from an antiproliferative mechanism in cancer (oncogene-induced senescence) to a critical component of physiological processes associated with embryonic development, tissue regeneration, ageing and its associated diseases. In somatic cells, oncogenic mutations in RAS-MAPK pathway genes are associated with oncogene-induced senescence and cancer, while germline mutations in the same pathway are linked to a group of monogenic developmental disorders generally termed RASopathies. Here, we consider that in these disorders, senescence induction may result in opposing outcomes, a tumour protective effect and a possible contributor to a premature ageing phenotype identified in Costello syndrome, which belongs to the RASopathy group. In this review, we will highlight the role of senescence in organismal homeostasis and we will describe the current knowledge about senescence in RASopathies. Additionally, we provide a perspective on examples of experimentally characterised RASopathy mutations that, alone or in combination with various stressors, may also trigger an age-dependent chronic senescence, possibly contributing to the age-dependent worsening of RASopathy pathophenotype and the reduction of lifespan.

DGAT1 mutations leading to delayed chronic diarrhoea: a case report.

BACKGROUND: Early-onset chronic diarrhoea often indicates a congenital disorder. Mutation in diacylglycerol o-acyltransferase 1 (DGAT1) has recently been linked to early-onset chronic diarrhoea. To date, only a few cases of DGAT1 deficiency have been reported. Diarrhoea in those cases was severe and developed in the neonatal period or within 2 months after birth. CASE PRESENTATION: Here, we report a female patient with DGAT1 mutations with delayed-onset chronic diarrhoea. The patient had vomiting, hypoalbuminemia, hypertriglyceridemia, and failure to thrive at early infancy. Her intractable chronic diarrhoea occurred until she was 8 months of age. A compound heterozygous DGAT1 mutation was found in the patient, which was first found in the Chinese population. Her symptoms and nutrition status improved after nutritional therapy, including a fat restriction diet. CONCLUSIONS: This case expanded our knowledge of the clinical features of patients with DGAT1 mutations. Intractable diarrhoea with delayed onset could also be a congenital disorder.

Cardio-facio-cutaneous syndrome-associated pathogenic MAP2K1 variants activate autophagy.

MAP2K1 encodes mitogen-activated protein kinase 1 (MEK1). Mutations in MAP2K1 lead to continuous activation of MEK/ERK signaling pathway, giving rise to cardio-facio-cutaneous syndrome (CFCS). However, the molecular mechanisms of abnormal activation of MEK/ERK signaling pathway and the role of autophagy, if any, in manifesting CFCS in MAP2K mutants remain unclear. Here, we report three Chinese children with CFCS having MAP2K1 pathogenic variants, identified by exome sequencing. They presented with dysmorphic facial features, seizures, psychomotor retardation, and short stature. Additionally, the third child showed pulmonary valve stenosis, multiple skeletal deformities, and osteoporosis. Whole exome sequencing revealed two heterozygous missense mutations in exon 3 of MAP2K1 (c.383G>T; p.Gly128Val and c.389A>G; p.Tyr130Cys), as well as a novel heterozygous missense variant (c.170A>T; p.Lys57Met) in exon 2 of MAP2K1. In SH-SY5Y cells, we identified, for the first time, that MAP2K1 mutations can activate the p-ERK-dependent cell cycle progression and autophagy, and cause CFCS. Our results extended the mutational spectrum of MAP2K1, examined the role of MEK1 protein in nerve cell functions, and demonstrated, for the first time, that autophagy may mediate the altered MAP2K1 function, leading to CFCS phenotypes.

A combination of two novel VARS2 variants causes a mitochondrial disorder associated with failure to thrive and pulmonary hypertension.

The VARS2 gene encodes a mitochondrial valyl-transfer RNA synthetase which is used in mitochondrial translation. To date, several patients with VARS2 pathogenic variants have been described in the literature. These patients have features of lactic acidosis with encephalomyopathy. We present a case of an infant with lactic acidosis, failure to thrive, and severe primary pulmonary hypertension who was found to be a compound heterozygote for two novel VARS2 variants (c.1940C>T, p.(Thr647Met) and c.2318G>A, p.(Arg773Gln)). The patient was treated with vitamin supplements and a carbohydrate-restricted diet. The lactic acidosis and failure to thrive resolved, and he showed good growth and development. Functional studies and molecular analysis employed a yeast model system and the VAS1 gene (yeast homolog of VARS2). VAS1 genes harboring either one of two mutations corresponding to the two novel variants in the VARS2 gene, exhibited partially reduced function in haploid yeast strains. A combination of both VAS1 variant alleles in a diploid yeast cell exhibited a more significant decrease in oxidative metabolism-dependent growth and in the oxygen consumption rate (reminiscent of the patient who carries two mutant VARS2 alleles). Our results demonstrate the pathogenicity of the biallellic novel VARS2 variants. KEY MESSAGES: • A case of an infant who is a compound heterozygote for two novel VARS2 variants. • This infant displayed lactic acidosis, failure to thrive, and pulmonary hypertension. • Treatment of the patient with a carbohydrate-restricted diet resulted in good growth and development. • Studies with the homologous yeast VAS1 gene showed reduced function of corresponding single mutant in haploid yeast strains. • A combination of both VAS1 variant alleles in diploid yeast exhibited a more significant decrease in function, thereby confirming the pathogenicity of the biallellic novel VARS2 variants.

C-type natriuretic peptide improves growth retardation in a mouse model of cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome, a genetic disorder caused by germline mutations in BRAF, KRAS, MAP2K1 and MAP2K2, is characterized by growth retardation, heart defects, dysmorphic facial appearance and dermatologic abnormalities. We have previously reported that knock-in mice expressing the CFC syndrome-associated mutation, Braf Q241R, showed growth retardation because of gastrointestinal dysfunction. However, other factors associated with growth retardation, including chondrogenesis and endocrinological profile, have not been examined. Here, we show that 3- and 4-week-old BrafQ241R/+ mice have decreased body weight and length, as well as reduced growth plate width in the proximal tibiae. Furthermore, proliferative and hypertrophic chondrocyte zones of the growth plate were reduced in BrafQ241R/+ mice compared with Braf+/+ mice. Immunohistological analysis revealed that extracellular signal-regulated kinase (ERK) activation was enhanced in hypertrophic chondrocytes in BrafQ241R/+ mice. In accordance with growth retardation and reduced growth plate width, decreased serum levels of insulin-like growth factor 1 (IGF-1) and IGF binding protein 3 (IGFBP-3) were observed in BrafQ241R/+ mice at 3 and 4 weeks of age. Treatment with C-type natriuretic peptide (CNP), a stimulator of endochondral bone growth and a potent inhibitor of the FGFR3-RAF1-MEK/ERK signaling, increased body and tail lengths in Braf+/+ and BrafQ241R/+ mice. In conclusion, ERK activation in chondrocytes and low serum IGF-1/IGFBP-3 levels could be associated with the growth retardation observed in BrafQ241R/+ mice. Our data also suggest that CNP is a potential therapeutic target in CFC syndrome.

Energy Intake and Expenditure in Children With Heart Failure.

OBJECTIVES: Up to one third of children with heart failure exhibit growth failure. Limited data exists reporting energy requirements in this population. A study was designed to characterize the energy intake and total daily energy expenditure of children with heart failure using the doubly labeled water method. DESIGN: Prospective study using doubly labeled water to measure total daily energy expenditure in children with heart failure. Doubly labeled water was administered orally and daily urine samples collected for 10 days. Total daily energy expenditure was compared with historic data from age- and gender-matched healthy population. Anthropometrics and 3-day calorie count were also done. SETTING: The Cardiovascular Intensive Care Unit and Cardiology ward at Texas Children's Hospital. PATIENTS: Children with new presentation of heart failure as defined by an ejection fraction less than 35% and requiring inotrope(s) at the time of enrollment. MEASUREMENTS AND MAIN RESULTS: A total of five children with heart failure were enrolled from 2015 to 2016. All children showed weight-for-length less than mean-for-age. All had depressed myocardial function at enrollment, and all but one demonstrated improvement in ejection fraction at follow-up. Three had energy intake that met or surpassed their total daily energy expenditure, with total daily energy expenditure that measured below historic controls. One infant, despite supplementation, had an energy intake substantially below that of measured total daily energy expenditure and required cardiac transplantation. CONCLUSIONS: Growth failure in heart failure is likely multifactorial and may be related to suboptimal energy intake secondary to exercise intolerance, malabsorption, and/or end-organ dysfunction due to impaired cardiac output. Doubly labeled water is a feasible method to assess total daily energy expenditure in children with heart failure.

Patient-derived iPSCs show premature neural differentiation and neuron type-specific phenotypes relevant to neurodevelopment.

Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAFQ257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events.

Impaired Osteogenesis of Disease-Specific Induced Pluripotent Stem Cells Derived from a CFC Syndrome Patient.

Cardiofaciocutaneous (CFC) syndrome is a rare genetic disorder caused by mutations in the extracellular signal-regulated kinase (ERK) signaling. However, little is known about how aberrant ERK signaling is associated with the defective bone development manifested in most CFC syndrome patients. In this study, induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts of a CFC syndrome patient having rapidly accelerated fibrosarcoma kinase B (BRAF) gain-of-function mutation. CFC-iPSCs were differentiated into mesenchymal stem cells (CFC-MSCs) and further induced to osteoblasts in vitro. The osteogenic defects of CFC-MSCs were revealed by alkaline phosphatase activity assay, mineralization assay, quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting. Osteogenesis of CFC-MSCs was attenuated compared to wild-type (WT)-MSCs. In addition to activated ERK signaling, increased p-SMAD2 and decreased p-SMAD1 were observed in CFC-MSCs during osteogenesis. The defective osteogenesis of CFC-MSCs was rescued by inhibition of ERK signaling and SMAD2 signaling or activation of SMAD1 signaling. Importantly, activation of ERK signaling and SMAD2 signaling or inhibition of SMAD1 signaling recapitulated the impaired osteogenesis in WT-MSCs. Our findings indicate that SMAD2 signaling and SMAD1 signaling as well as ERK signaling are responsible for defective early bone development in CFC syndrome, providing a novel insight on the pathological mechanism and therapeutic targets.

Deletion of ß1-integrin in collecting duct principal cells leads to tubular injury and renal medullary fibrosis.

The renal collecting duct (CD) contains two major cell types, intercalated (ICs) and principal cells (PCs). A previous report showed that deletion of ß1-integrin in the entire renal CD causes defective CD morphogenesis resulting in kidney dysfunction. However, subsequent deletion of ß1-integrin specifically in ICs and PCs, respectively, did not cause any morphological defects in the CDs. The discrepancy between these studies prompts us to reinvestigate the role of ß1-integrin in CD cells, specifically in the PCs. We conditionally deleted ß1-integrin in mouse CD PCs using a specific aquaporin-2 (AQP2) promoter Cre-LoxP system. The resulting mutant mice, ß-1f/fAQP2-Cre+, had lower body weight, failed to thrive, and died around 8-12 wk. Their CD tubules were dilated, and some of them contained cellular debris. Increased apoptosis and proliferation of PCs were observed in the dilated CDs. Trichrome staining and electron microscopy revealed the presence of peritubular and interstitial fibrosis that is associated with increased production of extracellular matrix proteins including collagen type IV and fibronectin, as detected by immunoblotting. Further analysis revealed a significantly increased expression of transforming growth factor-ß (TGF-ß)-induced protein, fibronectin, and TGF-ß receptor-1 mRNAs and concomitantly increased phosphorylation of SMAD-2 that indicates the activation of the TGF-ß signaling pathway. Therefore, our data reveal that normal expression of ß1-integrin in PCs is a critical determinant of CD structural and functional integrity and further support the previously reported critical role of ß1-integrin in the development and/or maintenance of the CD structure and function.

Serum and Hair Mineral Levels in Children with Failure to Thrive According to the Type of Feeding Difficulties.

This study evaluated serum and hair mineral and trace element levels as well as levels of other nutritional factors affecting growth and appetite in young children with non-organic failure to thrive (NOFTT) based on the presence or types of feeding difficulty (FD). Between August 2012 and July 2015, 136 children less than 6 years of age with NOFTT were included. FD was diagnosed based on Wolfson criteria and divided into subtypes according to Chatoor's classification. Clinical data were reviewed, and serum and hair mineral levels were measured. Of all assessed serum and hair minerals, only hair sulfur contents differed significantly between subjects with and without infantile anorexia (39,392 ± 2211 vs. 40,332 ± 2551 µg/g, P = 0.034). There were no differences in serum and hair mineral levels between children with and without sensory food aversion. Hair copper contents were significantly lower in children with FD of reciprocity (12.3 ± 6.0 vs. 22.4 ± 25.1 µg/g, P = 0.049). While hair zinc contents were also lower, the difference was not statistically significant (49.2 ± 26.8 vs. 70.6 ± 41.0 µg/g, P = 0.055). Only hair manganese contents were significantly lower in children with posttraumatic FD (0.12 ± 0.04 vs. 0.26 ± 0.73 µg/g, P = 0.037). In conclusion, there were no differences in most serum and hair mineral levels in children with NOFTT, except for relatively lower hair levels of sulfur, copper and possibly zinc, and manganese in infantile anorexia, reciprocity, and posttraumatic types of FD, respectively.

Biochemical and molecular characteristics of citrin deficiency in Korean children.

Mutations in SLC25A13 cause citrin deficiency, which has three phenotypes: neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD) and adult-onset type 2 citrullinemia (CTLN2). The purpose of this study was to determine the mutation spectrum and the clinical and biochemical characteristics of citrin deficiency in Korean patients. Thirty-four patients were diagnosed with citrin deficiency based on mutations in SLC25A13, as verified by direct sequencing and long PCR screening of a large transposon insertion. A total of 66 alleles from 33 unrelated families of 34 patients with citrin deficiency (27 NICCD, 2 FTTDCD and 5 CTLN2) were retrospectively identified. The common pathogenic alleles were IVS16ins3kb (33%), c.851_854del (30%) and c.1177+1G>A (12%), and three novel variants were identified. Levels of citrulline, threonine, methionine, tyrosine and arginine and the threonine-to-serine ratio were higher in children with neonatal intrahepatic cholestasis caused by NICCD compared with that in patients with idiopathic neonatal hepatitis (INH). We concluded that Korean patients with citrin deficiency showed the highest frequency of the IVS16ins3kb mutation and that plasma amino-acid profiles can be used to differentiate between NICCD and INH.

Objective studies of the face of Noonan, Cardio-facio-cutaneous, and Costello syndromes: A comparison of three disorders of the Ras/MAPK signaling pathway.

Noonan, Cardio-facio-cutaneous, and Costello syndromes are disorders of the Ras/MAPK pathway that share many clinical features. This observational and anthropometric study was conducted to describe the key facial features of each syndrome in order to improve discrimination between the three conditions, particularly in young children where diagnosis is most challenging. Direct measurement of the head and face was used to enhance diagnostic accuracy, and identify the most unusual or specific dimensions. The Noonan syndrome cohort included 123 individuals, aged 6 months to 41 years. There were 20 children and adolescents with Cardio-facio-cutaneous syndrome, and 28 individuals with Costello syndrome, aged 1-32 years. The facial phenotypes of these syndromes, particularly Noonan syndrome, are well-described but objective data have not been published in peer-reviewed literature. In this study, subjective observations, in the main, were validated by anthropometry with one exception. In individuals with Costello syndrome, mouth width was normal, thus the impression of wide mouth is likely due to full lips or the mouth being viewed in relation to a narrow lower face. When the three conditions were compared objectively, syndrome-specific pattern profiles showed high concordance in early life. At older ages, Cardio-facio-cutaneous syndrome was distinguished by increased width of the mid/lower face, and reduced growth of maxillary and mandibular dimensions was noted in both Noonan and Costello syndromes. Despite substantial similarities in face shape in older individuals with these two conditions, bulbous nasal tip, full lips, and an apparently wide mouth in those with Costello Syndrome facilitate discrimination from Noonan syndrome. © 2016 Wiley Periodicals, Inc.

Understanding Growth Failure in Costello Syndrome: Increased Resting Energy Expenditure.

Costello syndrome is a rare multisystem disorder caused by mutations in the proto-oncogene HRAS. Failure to thrive is one of its cardinal clinical features. This study documents that individuals with Costello syndrome have increased resting energy expenditure. We speculate this could be one of the potential mechanisms causing failure to thrive.

De novo dominant ASXL3 mutations alter H2A deubiquitination and transcription in Bainbridge-Ropers syndrome.

De novo truncating mutations in Additional sex combs-like 3 (ASXL3) have been identified in individuals with Bainbridge-Ropers syndrome (BRS), characterized by failure to thrive, global developmental delay, feeding problems, hypotonia, dysmorphic features, profound speech delays and intellectual disability. We identified three novel de novo heterozygous truncating variants distributed across ASXL3, outside the original cluster of ASXL3 mutations previously described for BRS. Primary skin fibroblasts established from a BRS patient were used to investigate the functional impact of pathogenic variants. ASXL3 mRNA transcripts from the mutated allele are prone to nonsense-mediated decay, and expression of ASXL3 is reduced. We found that ASXL3 interacts with BAP1, a hydrolase that removes mono-ubiquitin from histone H2A lysine 119 (H2AK119Ub1) as a component of the Polycomb repressive deubiquitination (PR-DUB) complex. A significant increase in H2AK119Ub1 was observed in ASXL3 patient fibroblasts, highlighting an important functional role for ASXL3 in PR-DUB mediated deubiquitination. Transcriptomes of ASXL3 patient and control fibroblasts were compared to investigate the impact of chromatin changes on transcriptional regulation. Out of 564 significantly differentially expressed genes (DEGs) in ASXL3 patient fibroblasts, 52% were upregulated and 48% downregulated. DEGs were enriched in molecular processes impacting transcriptional regulation, development and proliferation, consistent with the features of BRS. This is the first single gene disorder linked to defects in deubiquitination of H2AK119Ub1 and suggests an important role for dynamic regulation of H2A mono-ubiquitination in transcriptional regulation and the pathophysiology of BRS.

Enhanced SMAD1 Signaling Contributes to Impairments of Early Development in CFC-iPSCs.

Cardio-facio-cutaneous (CFC) syndrome is a developmental disorder caused by constitutively active ERK signaling manifesting mainly from BRAF mutations. Little is known about the role of elevated ERK signaling in CFC syndrome during early development. Here, we show that both SMAD1 and ERK signaling pathways may contribute to the developmental defects in CFC syndrome. Induced pluripotent stem cells (iPSCs) derived from dermal fibroblasts of a CFC syndrome patient (CFC-iPSCs) revealed early developmental defects in embryoid body (EB) development, ß-catenin localization, and neuronal differentiation. Both SMAD1 and ERK signalings were significantly activated in CFC-iPSCs during EB formation. Most of the ß-catenin was dissociated from the membrane and preferentially localized into the nucleus in CFC-EBs. Furthermore, activation of SMAD1 signaling recapitulated early developmental defects in wild-type iPSCs. Intriguingly, inhibition of SMAD1 signaling in CFC-iPSCs rescued aberrant EB morphology, impaired neuronal differentiation, and altered ß-catenin localization. These results suggest that SMAD1 signaling may be a key pathway contributing the pathogenesis of CFC syndrome during early development.

New BRAF knockin mice provide a pathogenetic mechanism of developmental defects and a therapeutic approach in cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome is one of the 'RASopathies', a group of phenotypically overlapping syndromes caused by germline mutations that encode components of the RAS-MAPK pathway. Germline mutations in BRAF cause CFC syndrome, which is characterized by heart defects, distinctive facial features and ectodermal abnormalities. To define the pathogenesis and to develop a potential therapeutic approach in CFC syndrome, we here generated new knockin mice (here Braf(Q241R/+)) expressing the Braf Q241R mutation, which corresponds to the most frequent mutation in CFC syndrome, Q257R. Braf(Q241R/+) mice manifested embryonic/neonatal lethality, showing liver necrosis, edema and craniofacial abnormalities. Histological analysis revealed multiple heart defects, including cardiomegaly, enlarged cardiac valves, ventricular noncompaction and ventricular septal defects. Braf(Q241R/+) embryos also showed massively distended jugular lymphatic sacs and subcutaneous lymphatic vessels, demonstrating lymphatic defects in RASopathy knockin mice for the first time. Prenatal treatment with a MEK inhibitor, PD0325901, rescued the embryonic lethality with amelioration of craniofacial abnormalities and edema in Braf(Q241R/+) embryos. Unexpectedly, one surviving pup was obtained after treatment with a histone 3 demethylase inhibitor, GSK-J4, or NCDM-32b. Combination treatment with PD0325901 and GSK-J4 further increased the rescue from embryonic lethality, ameliorating enlarged cardiac valves. These results suggest that our new Braf knockin mice recapitulate major features of RASopathies and that epigenetic modulation as well as the inhibition of the ERK pathway will be a potential therapeutic strategy for the treatment of CFC syndrome.

Tufting enteropathy revisited: the utility of MOC31 (EpCAM) immunohistochemistry in diagnosis.

Tufting enteropathy (TE) is an uncommon disease causing intractable diarrheas starting in early childhood and resulting in failure to thrive, dependence on total parenteral nutrition, and eventually requiring transplantation for treatment. The diagnosis has been based on histology showing the presence of epithelial "tufts" in the small bowel and colonic mucosa and variable villus alterations with mild to no inflammatory changes and preserved brush border. The gene for TE has been identified to be the EpCAM gene on chromosome 2p21. With Institutional Review Board approval, all cases of intractable diarrhea in children in whom TE was suspected or diagnosed were retrieved from the pathology files (17 patients). Other cases of infantile, neonatal, and childhood diarrhea were also retrieved to serve as controls for the staining studies (total 37 patients). EpCAM/MOC31 antibody staining was performed on all cases. The study cohort comprised 17 patients (13 boys, 4 girls) with a diagnosis of TE ranging in age at diagnosis from 3 months to 9 years, all presenting with protracted diarrhea and/or failure to thrive, usually since birth. Staining with MOC31 was carried out in all but 2 patients (both consults) and was completely negative in the epithelium irrespective of the site of biopsy or resection. In contrast, MOC31 was positive in all other cases tested, giving a sensitivity and specificity of 100% for loss of staining. MOC31 is a diagnostic stain for TE and should be included in the panel in any case of prolonged diarrhea in children to exclude this possibility.