Molecular and functional analysis of a novel MEK2 mutation in cardio-facio-cutaneous syndrome: transmission through four generations.

Cardio-facio-cutaneous (CFC) syndrome is one of the RASopathies and is caused by alteration of activity through the Ras/mitogen-activated protein kinase (MAPK) pathway due to heterozygous de novo mutations in protein kinases BRAF, MEK1, or MEK2. CFC is a rare multiple congenital anomaly disorder in which individuals have characteristic dysmorphic features, cardiac defects, ectodermal anomalies and developmental delay.We report a 7(1/2)-month-old boy with a clinical diagnosis of CFC. Bidirectional sequence analysis of MEK2 revealed a novel c.383C-->A transversion in exon 3 resulting in a nonsynonymous missense substitution, p.P128Q. Other family members, including the proband's mother and half-sibling, displayed phenotypic features of CFC and were also screened for the MEK2 mutation identified in the proband. Sorting Intolerant From Tolerant (SIFT) analysis determined the novel MEK2 p.P128Q to be deleterious. To corroborate the functional alteration of the novel mutant protein, transient transfection of HEK 293T cells with subsequent Western analysis was used to demonstrate increased kinase activity, as measured by ERK phosphorylation. This first reported case of a vertically transmitted functional CFC MEK mutation further expands our understanding of germline mutations within the Ras/MAPK pathway.

Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

The Ras/MAPK pathway is critical for human development and plays a central role in the formation and progression of most cancers. Children born with germ-line mutations in BRAF, MEK1 or MEK2 develop cardio-facio-cutaneous (CFC) syndrome, an autosomal dominant syndrome characterized by a distinctive facial appearance, heart defects, skin and hair abnormalities and mental retardation. CFC syndrome mutations in BRAF promote both kinase-activating and kinase-impaired variants. CFC syndrome has a progressive phenotype, and the availability of clinically active inhibitors of the MAPK pathway prompts the important question as to whether such inhibitors might be therapeutically effective in the treatment of CFC syndrome. To study the developmental effects of CFC mutant alleles in vivo, we have expressed a panel of 28 BRAF and MEK alleles in zebrafish embryos to assess the function of human disease alleles and available chemical inhibitors of this pathway. We find that both kinase-activating and kinase-impaired CFC mutant alleles promote the equivalent developmental outcome when expressed during early development and that treatment of CFC-zebrafish embryos with inhibitors of the FGF-MAPK pathway can restore normal early development. Importantly, we find a developmental window in which treatment with a MEK inhibitor can restore the normal early development of the embryo, without the additional, unwanted developmental effects of the drug.

Mutation analysis of BRAF, MEK1 and MEK2 in 15 ovarian cancer cell lines: implications for therapy.

BACKGROUND: Among gynecologic cancers, ovarian cancer is the second most common and has the highest death rate. Cancer is a genetic disorder and arises due to the accumulation of somatic mutations in critical genes. An understanding of the genetic basis of ovarian cancer has implications both for early detection and for therapeutic intervention in this population of patients. METHODOLOGY/PRINCIPAL FINDINGS: Fifteen ovarian cancer cell lines, commonly used for in vitro experiments, were screened for mutations using bidirectional direct sequencing in all coding regions of BRAF, MEK1 and MEK2. BRAF mutations were identified in four of the fifteen ovarian cancer cell lines studied. Together, these four cell lines contained four different BRAF mutations, two of which were novel. ES-2 had the common B-Raf p.V600E mutation in exon 15 and Hey contained an exon 11 missense mutation, p.G464E. The two novel B-Raf mutants identified were a 5 amino acid heterozygous deletion p.N486-P490del in OV90, and an exon 4 missense substitution p.Q201H in OVCAR 10. One of the cell lines, ES-2, contained a mutation in MEK1, specifically, a novel heterozygous missense substitution, p.D67N which resulted from a nt 199 G-->A transition. None of the cell lines contained coding region mutations in MEK2. Functional characterization of the MEK1 mutant p.D67N by transient transfection with subsequent Western blot analysis demonstrated increased ERK phosphorylation as compared to controls. CONCLUSIONS/SIGNIFICANCE: In this study, we report novel BRAF mutations in exon 4 and exon 12 and also report the first mutation in MEK1 associated with human cancer. Functional data indicate the MEK1 mutation may confer alteration of activation through the MAPK pathway. The significance of these findings is that BRAF and MEK1/2 mutations may be more common than anticipated in ovarian cancer which could have important implications for treatment of patients with this disease and suggests potential new therapeutic avenues.

Identification of a robust gene signature that predicts breast cancer outcome in independent data sets.

BACKGROUND: Breast cancer is a heterogeneous disease, presenting with a wide range of histologic, clinical, and genetic features. Microarray technology has shown promise in predicting outcome in these patients. METHODS: We profiled 162 breast tumors using expression microarrays to stratify tumors based on gene expression. A subset of 55 tumors with extensive follow-up was used to identify gene sets that predicted outcome. The predictive gene set was further tested in previously published data sets. RESULTS: We used different statistical methods to identify three gene sets associated with disease free survival. A fourth gene set, consisting of 21 genes in common to all three sets, also had the ability to predict patient outcome. To validate the predictive utility of this derived gene set, it was tested in two published data sets from other groups. This gene set resulted in significant separation of patients on the basis of survival in these data sets, correctly predicting outcome in 62-65% of patients. By comparing outcome prediction within subgroups based on ER status, grade, and nodal status, we found that our gene set was most effective in predicting outcome in ER positive and node negative tumors. CONCLUSION: This robust gene selection with extensive validation has identified a predictive gene set that may have clinical utility for outcome prediction in breast cancer patients.

Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder involving characteristic craniofacial features, cardiac defects, ectodermal abnormalities, and developmental delay. We demonstrate that heterogeneous de novo missense mutations in three genes within the mitogen-activated protein kinase (MAPK) pathway cause CFC syndrome. The majority of cases (18 out of 23) are caused by mutations in BRAF, a gene frequently mutated in cancer. Of the 11 mutations identified, two result in amino acid substitutions that occur in tumors, but most are unique and suggest previously unknown mechanisms of B-Raf activation. Furthermore, three of five individuals without BRAF mutations had missense mutations in either MEK1 or MEK2, downstream effectors of B-Raf. Our findings highlight the involvement of the MAPK pathway in human development and will provide a molecular diagnosis of CFC syndrome.

HRAS mutations in Costello syndrome: detection of constitutional activating mutations in codon 12 and 13 and loss of wild-type allele in malignancy.

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. Based on similarities with other cancer syndromes, we previously hypothesized that CS is likely due to activation of signal transduction through the Ras/MAPK pathway [Tartaglia et al., 2003]. In this study, the HRAS coding region was sequenced for mutations in a large, well-characterized cohort of 36 CS patients. Heterogeneous missense point mutations predicting an amino acid substitution were identified in 33/36 (92%) patients. The majority (91%) had a 34G --> A transition in codon 12. Less frequent mutations included 35G --> C (codon 12) and 37G --> T (codon 13). Parental samples did not have an HRAS mutation supporting the hypothesis of de novo heterogeneous mutations. There is phenotypic variability among patients with a 34G --> A transition. The most consistent features included characteristic facies and skin, failure to thrive, developmental delay, musculoskeletal abnormalities, visual impairment, cardiac abnormalities, and generalized hyperpigmentation. The two patients with 35G --> C had cardiac arrhythmias whereas one patient with a 37G --> T transversion had an enlarged aortic root. Of the patients with a clinical diagnosis of CS, neoplasia was the most consistent phenotypic feature for predicating an HRAS mutation. To gain an understanding of the relationship between constitutional HRAS mutations and malignancy, HRAS was sequenced in an advanced biphasic rhabdomyosarcoma/fibrosarcoma from an individual with a 34G --> A mutation. Loss of the wild-type HRAS allele was observed, suggesting tumorigenesis in CS patients is accompanied by additional somatic changes affecting HRAS. Finally, due to phenotypic overlap between CS and cardio-facio-cutaneous (CFC) syndromes, the HRAS coding region was sequenced in a well-characterized CFC cohort. No mutations were found which support a distinct genetic etiology between CS and CFC syndromes.

Differentiation of lobular versus ductal breast carcinomas by expression microarray analysis.

Invasive lobular and ductal breast tumors have distinct histologies and clinical presentation. Other than altered expression of E-cadherin, little is known about the underlying biology that distinguishes the tumor subtypes. We used cDNA microarrays to identify genes differentially expressed between lobular and ductal tumors. Unsupervised clustering of tumors failed to distinguish between the two subtypes. Prediction analysis for microarrays (PAM) was able to predict tumor type with an accuracy of 93.7%. Genes that were significantly differentially expressed between the two groups were identified by MaxT permutation analysis using t tests (20 cDNA clones and 10 unique genes), significance analysis for microarrays (33 cDNA clones and 15 genes, at an estimated false discovery rate of 2%), and PAM (31 cDNAs and 15 genes). There were 8 genes identified by all three of these related methods (E-cadherin, survivin, cathepsin B, TPI1, SPRY1, SCYA14, TFAP2B, and thrombospondin 4), and an additional 3 that were identified by significance analysis for microarrays and PAM (osteopontin, HLA-G, and CHC1). To validate the differential expression of these genes, 7 of them were tested by real-time quantitative PCR, which verified that they were differentially expressed in lobular versus ductal tumors. In conclusion, specific changes in gene expression distinguish lobular from ductal breast carcinomas. These genes may be important in understanding the basis of phenotypic differences among breast cancers.

Optimizing stringency for expression microarrays.

While several studies have reported methods to optimize expression microarray protocols, none have dealt directly with hybridization wash stringency. We designed a series of experiments to determine the optimal stringency conditions for microarray experiments, using reproducibility and magnitudes of log2 (test/reference) ratio values as measures of quality. Low-stringency wash conditions of cell line hybridizations led to nonspecific binding, resulting in increased intensities, decreased magnitude of ratios, and poor reproducibility. Relatively high-stringency wash conditions were found to give the best reproducibility and large magnitude ratio changes, although increasing the stringency beyond this point led to lower magnitude ratios and poorer reproducibility. The expression levels of the ERBB2 oncogene in the BT474 versus MCF7 cell lines showed that high-stringency wash conditions gave the best agreement with real-time quantitative PCR, although the magnitude of the changes by microarray was smaller than for real-time quantitative PCR. Analysis of a series of cell lines washed at the optimized stringency indicated that the rank order of relative expression levels for ERBB2 microarray clones agreed well with the rank order of ERBB2 levels, as measured by quantitative PCR. These results indicate that the optimization of stringency conditions will improve microarray reproducibility and give more representative expression values.