Comparison of hair manifestations in cardio-facio-cutaneous and Costello syndromes highlights the influence of the RAS pathway on hair growth.

BACKGROUND: Abnormal hair growth is a defining feature of RASopathies, syndromes caused by germline mutations in the RAS pathway. However, detailed hair manifestations and the mechanisms of altered hair growth in RASopathies are poorly delineated. OBJECTIVES: To identify distinguishing clinical features and investigate how the RAS pathway influences hair growth by performing a systematic and detailed side-by-side comparison of hair manifestations in cardio-facio-cutaneous syndrome (CFCS) and Costello syndrome (CS), two RASopathies caused by mutations in the downstream and upstream elements of the RAS pathway, respectively. METHODS: Sixteen individuals with CFCS and 23 individuals with CS were enrolled. Mutation data were recorded. Scalp hair, eyebrows and eyelashes of individuals with CFCS or CS were examined for texture, colour, density and morphology. Scalp hairs were examined by light microscopy. RESULTS: While both syndromes displayed abnormal hair, striking differences were observed, including darker and thicker scalp hair and sparse eyebrows and eyelashes in CFCS. By contrast, synophrys, trichomegaly and abnormalities of the scalp hair shafts were observed in CS. Possible correlation with straight hair and genotype was observed in CS. CONCLUSION: The results emphasize the role of the RAS pathway in hair growth, improve accuracy of clinical diagnosis of CFCS and CS and provide a foundation for identification of therapeutic targets.

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.

Deletion of MAP2K2/MEK2: a novel mechanism for a RASopathy?

RASopathies are a class of genetic syndromes caused by germline mutations in genes encoding Ras/mitogen-activated protein kinase (Ras/MAPK) pathway components. Cardio-facio-cutaneous (CFC) syndrome is a RASopathy characterized by distinctive craniofacial features, skin and hair abnormalities, and congenital heart defects caused by activating mutations of BRAF, MEK1, MEK2, and KRAS. We define the phenotype of seven patients with de novo deletions of chromosome 19p13.3 including MEK2; they present with a distinct phenotype but have overlapping features with CFC syndrome. Phenotypic features of all seven patients include tall forehead, thick nasal tip, underdeveloped cheekbones, long midface, sinuous upper vermilion border, tall chin, angular jaw, and facial asymmetry. Patients also have developmental delay, hypotonia, heart abnormalities, failure to thrive, obstructive sleep apnea, gastroesophageal reflux and integument abnormalities. Analysis of epidermal growth factor-stimulated fibroblasts revealed that P-MEK1/2 was ∼50% less abundant in cells carrying the MEK2 deletion compared to the control. Significant differences in total MEK2 and Sprouty1 abundance were also observed. Our cohort of seven individuals with MEK2 deletions has overlapping features associated with RASopathies. This is the first report suggesting that, in addition to activating mutations, MEK2 haploinsufficiency can lead to dysregulation of the MAPK pathway.

Craniofacial and dental development in cardio-facio-cutaneous syndrome: the importance of Ras signaling homeostasis.

Cardio-facio-cutaneous syndrome (CFC) is a RASopathy that is characterized by craniofacial, dermatologic, gastrointestinal, ocular, cardiac, and neurologic anomalies. CFC is caused by activating mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathway that is downstream of receptor tyrosine kinase (RTK) signaling. RTK signaling is known to play a central role in craniofacial and dental development, but to date, no studies have systematically examined individuals with CFC to define key craniofacial and dental features. To fill this critical gap in our knowledge, we evaluated the craniofacial and dental phenotype of a large cohort (n = 32) of CFC individuals who attended the 2009 and 2011 CFC International Family Conferences. We quantified common craniofacial features in CFC which include macrocephaly, bitemporal narrowing, convex facial profile, and hypoplastic supraorbital ridges. In addition, there is a characteristic dental phenotype in CFC syndrome that includes malocclusion with open bite, posterior crossbite, and a high-arched palate. This thorough evaluation of the craniofacial and dental phenotype in CFC individuals provides a step forward in our understanding of the role of RTK/MAPK signaling in human craniofacial development and will aid clinicians who treat patients with CFC.

Dermatological phenotype in Costello syndrome: consequences of Ras dysregulation in development.

BACKGROUND: The RASopathies are a class of human genetic syndromes caused by germline mutations in genes that encode protein components of the Ras/mitogen-activated protein kinase (MAPK) pathway. Costello syndrome (CS) is a RASopathy caused by mutations in the HRAS gene, a key regulator of signal transduction. OBJECTIVE: To quantify the specific cutaneous phenotype observed in 46 individuals with Costello syndrome with confirmed HRAS mutations. METHODS: This was a cross-sectional study. Dermatological surveys were designed by the authors and were completed by parents of mutation-positive individuals with CS at the Costello Syndrome Family Network (CSFN) conferences in 2007 and 2009. Dermatological examinations were performed by the authors at the CSFN conferences. RESULTS: Cutaneous papillomas were reported in 33 of the 46 (72%) participants, with age of onset ranging from infancy to 22years. Individuals with CS are more likely than patients with cardiofaciocutaneous syndrome (CFC) to present with cutaneous papillomas (72% vs. 5%, P<0·001) and palmoplantar keratoderma (76% vs. 36%, P<0·001). Individuals with CS are less likely than individuals with CFC to present with sparse or absent eyebrows (9% vs. 90%, P<0·001) or keratosis pilaris (33% vs. 80%, P=0·001). This study also identified that loose, redundant skin on the hands and feet, 'stippled' dermatoglyphs (pachydermatoglyphia) on the fingertips (eight of 26, 31%) and acanthosis nigricans (17 of 46, 37%) are frequent features of CS. CONCLUSIONS: While there is significant phenotypic overlap among syndromes of the Ras/MAPK pathway, individuals with CS are more likely than individuals with CFC syndrome to present with cutaneous papillomas, palmoplantar keratoderma and full eyebrows, and are less likely to present with ulerythema ophryogenes, keratosis pilaris or multiple naevi. The dermatological features of CS, a Ras dysregulation syndrome, share many features with cutaneous paraneoplastic syndromes. This may provide further insight into the role of Ras signalling in cutaneous paraneoplastic syndromes.

Congenital diaphragmatic hernia in Smith-Magenis syndrome: a possible locus at chromosome 17p11.2.

We report on a 7-month-old girl with Smith-Magenis syndrome (SMS) due to a 4.76-Mb deletion of 17p12-17p11.2 detected by array comparative genomic hybridization. She was also affected with a left-sided congenital diaphragmatic hernia (CDH) and cardiac anomalies including an atypical atrioventricular canal defect and a cleft mitral valve. To our knowledge, this is the first reported case of a patient with both SMS and CDH. There are numerous chromosomal regions in which duplications, deletions, inversions, or translocations have been associated with CDH, but none have previously been reported at or close to 17p11.2. We discuss candidate genes for the diaphragmatic defect in this patient. Our case demonstrates that it is important to consider the possibility of SMS in non-isolated cases of diaphragmatic hernia.

Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome.

BRAF, the protein product of BRAF, is a serine/threonine protein kinase and one of the direct downstream effectors of Ras. Somatic mutations in BRAF occur in numerous human cancers, whereas germline BRAF mutations cause cardio-facio-cutaneous (CFC) syndrome. One recurrent somatic mutation, p.V600E, is frequently found in several tumor types, such as melanoma, papillary thyroid carcinoma, colon cancer, and ovarian cancer. However, a germline mutation affecting codon 600 has never been described. Here, we present a patient with CFC syndrome and a de novo germline mutation involving codon 600 of BRAF, thus providing the first evidence that a pathogenic germline mutation involving this critical codon is not only compatible with development but can also cause the CFC phenotype. In vitro functional analysis shows that this mutation, which replaces a valine with a glycine at codon 600 (p.V600G), leads to increased ERK and ELK phosphorylation compared to wild-type BRAF but is less strongly activating than the cancer-associated p.V600E mutation.

Dermatological findings in 61 mutation-positive individuals with cardiofaciocutaneous syndrome.

BACKGROUND: The RASopathies are a class of human genetic syndromes that are caused by germline mutations in genes which encode components of the Ras/mitogen-activated protein kinase (MAPK) pathway. Cardiofaciocutaneous (CFC) syndrome is characterized by distinctive craniofacial features, congenital heart defects, and abnormalities of the skin and hair. OBJECTIVES: Systematically to characterize the spectrum of dermatological findings in mutation-positive individuals with CFC syndrome. METHODS: Dermatological surveys were designed by the authors and distributed to the study participants through CFC International or directly by the authors (K.A.R. and D.H.S.) between July 2006 and August 2009. A second follow-up survey was collected between December 2007 and August 2009. When available, digital images and medical records of the participants were obtained. Study participants included individuals with CFC syndrome who have a mutation in BRAF, MAP2K1, MAP2K2 or KRAS. RESULTS: Individuals with CFC syndrome have a variety of dermatological manifestations caused by dysregulation of the MAPK pathway in development. Numerous acquired melanocytic naevi were one of the most striking features: more than 50 naevi were reported by 23% (14/61) of participants and of those, more than 100 naevi were reported by 36% (5/14). Keratosis pilaris was reported in 80% (49/61) of cases. Ulerythema ophryogenes was common, occurring in 90% (55/61). Infantile haemangiomas occurred at a greater frequency, 26% (16/61), as compared with the general population. CONCLUSIONS: CFC syndrome has a complex dermatological phenotype with many cutaneous features, some of which allow it to be differentiated from the other Ras/MAPK pathway syndromes. Multiple café-au-lait macules and papillomas were not identified in this CFC cohort, helping to distinguish CFC from other RASopathies such as neurofibromatosis type 1 and Costello syndrome.

Interstitial deletions of chromosome 6q: genotype-phenotype correlation utilizing array CGH.

Interstitial deletions of the long arm of chromosome 6 are relatively rare, with fewer than 100 cases reported. Phenotypic variation is in large part due to differences in size and location of the segmental aneuploidy. We report three new patients with interstitial deletions of chromosome 6q defined at the molecular level by array comparative genomic hybridization (array CGH). In two of three cases, the molecular breakpoints differed from those indicated by conventional karyotyping, demonstrating the enhanced resolution of array CGH. Two patients had minimal deletions of 6 and 8.8 Mb involving 6q16.2-->q21, and the third patient had a deletion of 11.3 Mb spanning 6q15-->q21. All three had developmental delay, craniofacial dysmorphology, and functional eye disorders, suggesting that genes affecting brain and craniofacial development are located in 6q16.2-->q21, the deleted region common to all three patients. Furthermore, gene(s) for discordant phenotypic features, such as central diabetes insipidus, may reside at 6q15, the monosomic region unique to patient 3. All three cases described here showed loss of paternal alleles within the deleted segment, providing further evidence of the predominantly paternal origin for 6q deletions and rearrangements.

HRAS and the Costello syndrome.

Costello syndrome (CS) is a complex developmental disorder involving characteristic craniofacial features, failure to thrive, developmental delay, cardiac and skeletal anomalies and a predisposition to develop neoplasia, both benign and malignant. CS is caused by activating germline mutations in HRAS and belongs to an exciting class of genetic syndromes that are caused by perturbation of function through the Ras pathway. Some of these other syndromes include Noonan syndrome, LEOPARD syndrome, neurofibromatosis 1 and cardio-facio-cutaneous syndrome. Ras is a critical signaling hub in the cell and is activated by receptor tyrosine kinases, G-protein-coupled receptors, cytokine receptors and extracellular matrix receptors. The downstream effectors of Ras are many and control vital cellular functions including cell cycle progression, cell survival, motility, transcription, translation and membrane trafficking. Understanding the genetic etiology of CS is the first step in gaining insight to the role Ras plays in human development, cellular signaling and cancer pathogenesis.

Expression of the coxsackievirus- and adenovirus receptor in gastrointestinal cancer correlates with tumor differentiation.

Modified adenoviruses represent a new approach to treatment of gastrointestinal cancer. However, their uptake by cells in many cases requires the major receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR). Thus, lack of CAR expression is a potential cause of intrinsic resistance of tumor cells to this type of treatment. To evaluate this, we studied the localization of CAR protein in normal and malignant gastrointestinal tissues. In normal tissues, CAR was concentrated at sites of cell-cell interaction, in particular at the apico-lateral cellular surface. Expression was particularly strong around bile and pancreatic ducts, which is in agreement with CAR's physiological function as a tight-junction protein. In GI malignancies (esophageal, pancreatic, colorectal and liver cancer), expression of the receptor varied substantially. Loss of CAR expression at cell-cell junction was evident in many samples. A significant correlation between CAR expression and histological grade was found, with moderately to poorly differentiated tumors most frequently demonstrating loss or reduction of CAR expression. These data indicate that CAR expression is frequently altered in gastrointestinal malignancy, potentially reducing the efficacy of adenovirus-based therapies.

Prader-Willi syndrome resulting from an unbalanced translocation: characterization by array comparative genomic hybridization.

Prader-Willi syndrome (PWS) is caused by lack of expression of paternally inherited genes on chromosome 15q11-->15q13. Most cases result from microdeletions in proximal chromosome 15q. The remainder results from maternal uniparental disomy of chromosome 15, imprinting center defects, and rarely from balanced or unbalanced chromosome rearrangements involving chromosome 15. We report a patient with multiple congenital anomalies, including craniofacial dysmorphology, microcephaly, bilateral cryptorchidism, and developmental delay. Cytogenetic analysis showed a de novo 45,XY,der(5)t(5;15)(p15.2;q13), -15 karyotype. In effect, the proband had monosomies of 5p15.2-->pter and 15pter-->15q13. Methylation polymerase chain reaction analysis of the promoter region of the SNRPN gene showed only the maternal allele, consistent with the PWS phenotype. The proband's expanded phenotype was similar to other patients who have PWS as a result of unbalanced translocations and likely reflects the contribution of the associated monosomy. Array comparative genomic hybridization (array CGH) confirmed deletions of both distal 5p and proximal 15q and provided more accurate information as to the size of the deletions and the molecular breakpoints. This case illustrates the utility of array CGH in characterizing complex constitutional structural chromosome abnormalities at the molecular level.

Exclusion of PTPN11 mutations in Costello syndrome: further evidence for distinct genetic etiologies for Noonan, cardio-facio-cutaneous and Costello syndromes.

Costello syndrome (CS) is a rare, multiple congenital anomaly syndrome with characteristic dysmorphic features, cardiac anomalies and a tendency to develop certain cancers. Phenotypically there is some overlap with other genetic disorders, notably cardio-facio-cutaneous (CFC) syndrome and Noonan syndrome (NS), suggesting that these syndromes may be allelic. We recently identified PTPN11, which encodes the non-receptor protein tyrosine phosphatase, SHP-2, as a major NS disease gene. In this report, we screened a cohort of 27 patients, with the clinical diagnosis of CS, for PTPN11 mutations using denaturing high performance liquid chromatography analysis. No mutations of the PTPN11 gene were found in the CS patients. Common polymorphisms in introns 6 and 7 and exon 8 were identified in four individuals. With our previous exclusion of PTPN11 mutations in CFC syndrome, these data suggest distinct genetic etiologies for Noonan, CFC and Costello syndromes.

Periventricular heterotopia associated with chromosome 5p anomalies.

Periventricular heterotopia (PH) is characterized by neuronal nodules along the lateral ventricles. Whereas mutations in X-linked FLNA cause such cortical malformations, the authors report two cases of PH localizing to chromosome 5p. Both subjects have complex partial seizures. MRI demonstrated bilateral nodular PH, with subcortical heterotopia or focal gliosis. FISH identified a duplication of 5p15.1 [46,XX,dup(5)(p15.1p15.1)] and a trisomy of 5p15.33 [46,XY,der(14)t(5;14)(p15.33;p11.2) mat]. These findings suggest a new PH locus along the telomeric end of chromosome 5p.

Disruption of 3D tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor.

The human coxsackievirus and adenovirus receptor (CAR) represents the primary cellular site of adenovirus attachment during infection. An understanding of the mechanisms regulating its expression could contribute to improving efficacy and safety of adenovirus-based therapies. We characterized regulation of CAR expression in a 3D cell culture model of human breast cancer progression, which mimics aspects of the physiological tissue context in vitro. Phenotypically normal breast epithelial cells (S1) and their malignant derivative (T4-2 cells) were grown either on tissue culture plastic (2D) or 3D cultures in basement membrane matrix. S1 cells grown in 3D showed low levels of CAR, which was expressed mainly at cell-cell junctions. In contrast, T4-2 cells expressed high levels of CAR, which was mainly in the cytoplasm. When signaling through the epidermal growth factor receptor was inhibited in T4-2 cells, cells reverted to a normal phenotype, CAR protein expression was significantly reduced, and the protein relocalized to cell-cell junctions. Growth of S1 cells as 2D cultures or in 3D in collagen-I, a nonphysiological microenvironment for these cells, led to up-regulation of CAR to levels similar to those in T4-2 cells, independently of cellular growth rates. Thus, expression of CAR depends on the integrity and polarity of the 3D organization of epithelial cells. Disruption of this organization by changes in the microenvironment, including malignant transformation, leads to up-regulation of CAR, thus enhancing the cell's susceptibility to adenovirus infection.

Tandem duplication mosaicism: characterization of a mosaic dup(5q) and review.

Mosaicism for tandem duplications is rare. Most patients reported had abnormal phenotypes of varying severity, depending on the chromosomal imbalance involved and the level of mosaicism. Post-zygotic unequal sister-chromatid exchange has been proposed as the main mechanism for tandem duplication mosaicism. However, previous molecular analyses have implicated both meiotic and post-zygotic origins for the duplication. We describe a newborn male who was originally diagnosed in utero with arrhythmia and tetralogy of Fallot. He had multiple dysmorphic features including telecanthus, blepharophimosis, high broad nasal bridge with a square-shaped nose, flat philtrum, thin upper lip, down-turned corners of the mouth, high-arched palate, micrognathia, asymmetric ears, and long, thin fingers and toes. Karyotyping of peripheral blood lymphocytes showed mosaicism for a tandem duplication of part of the long arm of one chromosome 5: mos46,XY,dup(5)(q13q33)[6]/46,XY[45]. Fibroblast cultures had the same mosaic karyotype with a higher frequency of the dup(5) clone: mos46,XY,dup(5)(q13q33)[9]/46,XY[21]. Fluorescence in situ hybridization analysis with a wcp5 confirmed the chromosome 5 origin of the additional material. Parental karyotypes were normal indicating a de novo origin of the dup(5) in the proband. Molecular analyses of chromosome 5 sequence-tagged-site (STS) markers in our family were consistent with a post-zygotic origin for the duplication. Therefore, mosaicism for tandem duplications can arise both through meiotic or mitotic errors, as a result of unequal crossing over or unequal sister-chromatid exchange, respectively. Our review indicates that mosaicism for tandem duplications is likely under-ascertained and that parental karyotyping of probands with non-mosaic tandem duplications should be performed.

Cardio-facio-cutaneous syndrome phenotype in an individual with an interstitial deletion of 12q: identification of a candidate region for CFC syndrome.

We report on a 19-month-old girl who presented with the phenotype of cardio-faciocutaneous (CFC) syndrome including characteristic minor facial anomalies, cardiac defect, ectodermal anomalies, and developmental delay. Cytogenetic analysis showed the presence of an interstitial deletion of one chromosome 12, del(12)(q21.2q22), confirmed by fluorescence in situ hybridization with chromosome band specific probes. Controversy exists as to whether CFC and Noonan syndrome (NS) are distinct disorders, a contiguous gene syndrome, or allelic variants. The identification of the del(12) in this patient, in a region distinct from the putative NS locus, supports the view that CFC is a genetically distinct condition from NS. In addition, this implicates the region 12q21.2-->4q22 as a candidate region for the gene(s) causing CFC syndrome.