Neurofibromin is engaged in many cellular processes and when the proper protein functioning is impaired, it causes neurofibromatosis type 1 (NF1), one of the most common inherited neurological disorders. Recent advances in sequencing and screening of the NF1 gene have increased the number of detected variants. However, the correlation of these variants with the clinic remains poorly understood. In this study, we analyzed 4610 germinal NF1 variants annotated in ClinVar and determined on exon level the mutational spectrum and potential pathogenic regions. Then, a binomial and sliding windows test using 783 benign and 938 pathogenic NF1 variants were analyzed against functional and structural regions of neurofibromin. The distribution of synonymous, missense, and frameshift variants are statistically significant in certain regions of neurofibromin suggesting that the type of variant and its associated phenotype may depend on protein disorder. Indeed, there is a negative correlation between the pathogenic fraction prediction and the disorder data, suggesting that the higher an intrinsically disordered region is, the lower the pathogenic fraction is and vice versa. Most pathogenic variants are associated to NF1 and our analysis suggests that GRD, CSRD, TBD, and Armadillo1 domains are hotspots in neurofibromin. Knowledge about NF1 genotype–phenotype correlations can provide prognostic guidance and aid in organ-specific surveillance.

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the transcription of the RNA polymerase III, and the processing of ribosomal RNA. Bud27 also regulates both RNA polymerase II-dependent transcription of ribosomal proteins and ribosome biogenesis regulon genes, likely by occupying their DNA ORFs, and the processing of the corresponding mRNAs. With RNA polymerase II, this association occurs in a transcription rate-dependent manner. Our data also indicate that Bud27 inactivation alters the phosphorylation kinetics of ribosomal protein S6, a readout of TORC1 activity. We conclude that Bud27 impacts the homeostasis of the ribosome biogenesis process by regulating the activity of the three RNA polymerases and, in this way, the synthesis of ribosomal components. This quite likely occurs through a functional connection of Bud27 with the TOR signaling pathway.

In recent years, high-throughput next-generation sequencing technology has allowed a rapid increase in diagnostic capacity and precision through different bioinformatics processing algorithms, tools and pipelines. The identification, annotation and classification of sequence variants within different target regions are now considered a gold standard in clinical genetic diagnosis. However, this procedure lacks the ability to link regulatory events such as differential splicing to diseases. RNA-seq is necessary in clinical routine in order to interpret and detect among others splicing events and splicing variants, as it would increase the diagnostic rate by up to 10-35%. The transcriptome has a very dynamic nature, varying according to tissue type, cellular conditions and environmental factors that may affect regulatory events such as splicing and the expression of genes or their isoforms. RNA-seq offers a robust technical analysis of this complexity, but it requires a profound knowledge of computational/statistical tools that may need to be adjusted depending on the disease under study. In this article we will cover RNA-seq analyses best practices applied to clinical routine, bioinformatics procedures and present challenges of this approach.

During neurogenesis, dynamic developmental cues, transcription factors and histone modifying enzymes regulate the gene expression programs by modulating the activity of neural-specific enhancers. How transient developmental signals coordinate transcription factor recruitment to enhancers and to which extent chromatin modifiers contribute to enhancer activity is starting to be uncovered. Here, we take advantage of neural stem cells as a model to unravel the mechanisms underlying neural enhancer activation in response to the TGFβ signaling. Genome-wide experiments demonstrate that the proneural factor ASCL1 assists SMAD3 in the binding to a subset of enhancers. Once located at the enhancers, SMAD3 recruits the histone demethylase JMJD3 and the remodeling factor CHD8, creating the appropriate chromatin landscape to allow enhancer transcription and posterior gene activation. Finally, to analyze the phenotypical traits owed to cis-regulatory regions, we use CRISPR-Cas9 technology to demonstrate that the TGFβ-responsive Neurog2 enhancer is essential for proper neuronal polarization.

A precise immune response is essential for cellular homeostasis and animal survival. The paramount importance of its control is reflected by the fact that its non-specific activation leads to inflammatory events that ultimately contribute to the appearance of many chronic diseases. However, the molecular mechanisms preventing non-specific activation and allowing a quick response upon signal activation are not yet fully understood. In this paper we uncover a new function of PHF8 blocking signal independent activation of immune gene promoters. Affinity purifications coupled with mass spectrometry analysis identified SIN3A and HDAC1 corepressors as new PHF8 interacting partners. Further molecular analysis demonstrated that prior to interferon gamma (IFNγ) stimulation, PHF8 is bound to a subset of IFNγ-responsive promoters. Through the association with HDAC1 and SIN3A, PHF8 keeps the promoters in a silent state, maintaining low levels of H4K20me1. Upon IFNγ treatment, PHF8 is phosphorylated by ERK2 and evicted from the promoters, correlating with an increase in H4K20me1 and transcriptional activation. Our data strongly indicate that in addition to its well-characterized function as a coactivator, PHF8 safeguards transcription to allow an accurate immune response.

Cancer cells exhibit multiple epigenetic changes with prominent local DNA hypermethylation and widespread hypomethylation affecting large chromosomal domains. Epigenome studies often disregard the study of repeat elements owing to technical complexity and their undefined role in genome regulation. We have developed NSUMA (Next-generation Sequencing of UnMethylated Alu), a cost-effective approach allowing the unambiguous interrogation of DNA methylation in more than 130,000 individual Alu elements, the most abundant retrotransposon in the human genome. DNA methylation profiles of Alu repeats have been analyzed in colon cancers and normal tissues using NSUMA and whole-genome bisulfite sequencing. Normal cells show a low proportion of unmethylated Alu (1%4%) that may increase up to 10-fold in cancer cells. In normal cells, unmethylated Alu elements tend to locate in the vicinity of functionally rich regions and display epigenetic features consistent with a direct impact on genome regulation. In cancer cells, Alu repeats are more resistant to hypomethylation than other retroelements. Genome segmentation based on high / low rates of Alu hypomethylation allows the identification of genomic compartments with differential genetic, epigenetic, and transcriptomic features. Alu hypomethylated regions show low transcriptional activity, late DNA replication, and its extent is associated with higher chromosomal instability. Our analysis demonstrates that Alu retroelements contribute to define the epigenetic landscape of normal and cancer cells and provides a unique resource on the epigenetic dynamics of a principal, but largely unexplored, component of the primate genome.

Antecedentes. La distrofia muscular cintura-cadera tipo 1B es una enfermedad con herencia autosómica dominante y secundaria a una mutación en el gen LMNA. Esta enfermedad se caracteriza por su afectación a nivel neuromuscular y cardiaco. Objetivo. Realizar diagnóstico clínico y confirmatorio molecular en una paciente con debilidad muscular proximal y sintomatología cardíaca a través de secuenciación exómica. Materiales y métodos. Se presenta el caso de una paciente de 57 años de edad con cuadro de debilidad muscular proximal progresiva principalmente en extremidades y posterior afectación cardíaca; adicionalmente, la paciente tiene múltiples familiares con la misma sintomatología. Se realizó estudio de secuenciación exómica con confirmación, por método de Sanger, de la mutación hallada y posteriormente el análisis bioinformático de esta. Resultados. La detección de la mutación R377L en el gen LMNA por secuenciación exómica con confirmación por Sanger, junto con la sintomatología clínica de la paciente y el análisis bioinformático de la mutación hallada, permitió realizar diagnóstico confirmatorio de distrofia muscular cintura-cadera tipo 1B. Conclusión. Es difícil realizar un diagnóstico clínico debido a la heterogeneidad genética del fenotipo de distrofias musculares cintura-cadera. La aproximación diagnóstica es compleja y requiere clasificar las distrofias musculares según el patrón de afectación y el patrón de herencia de la enfermedad. Adicionalmente, debido a los múltiples genes que pueden generar clínica semejante a las diferentes distrofias musculares, se recomienda realizar secuenciación exómica solicitando especial énfasis en los genes candidatos de distrofias musculares cintura-cadera.

The progressive restriction of differentiation potential from pluripotent embryonic stem cells (ESCs) to tissue-specific stem cells involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Skeletal muscle stem cells are required for the growth, maintenance, and regeneration of skeletal muscle. To investigate the contribution of DNA methylation to the establishment of the myogenic program, we analyzed ESCs, skeletal muscle stem cells in proliferating (myoblasts) and differentiating conditions (myotubes), and mature myofibers. About 1.000 differentially methylated regions were identified during muscle-lineage determination and terminal differentiation, mainly located in gene bodies and intergenic regions. As a whole, myogenic stem cells showed a gain of DNA methylation, while muscle differentiation was accompanied by loss of DNA methylation in CpG-poor regions. Notably, the hypomethylated regions in myogenic stem cells were neighbored by enhancer-type chromatin, suggesting the involvement of DNA methylation in the regulation of cell-type specific enhancers. Interestingly, we demonstrated the hypomethylation of the muscle cell-identity Myf5 super-enhancer only in muscle cells. Furthermore, we observed that upstream stimulatory factor 1 binding to Myf5 super-enhancer occurs upon DNA demethylation in myogenic stem cells. Taken altogether, we characterized the unique DNA methylation signature of skeletal muscle stem cells and highlighted the importance of DNA methylation-mediated regulation of cell identity Myf5 super-enhancer during cellular differentiation.

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Genetic characterization and monitoring of brain tumors is challenging given the restricted sampling of tumors and the limited abundance of brain tumor-derived circulating tumor DNA (ctDNA) in the plasma. Here, we sought to define whether cerebrospinal fluid (CSF) could serve as a ‘liquid biopsy’ for the genetic characterization of tumor DNA originated from the central nervous system (CNS) of patients (pts) with brain tumors. Methods: CSF, plasma and tumor tissue were obtained from pts with glioblastoma (GBM), brain metastases from breast (BMBC) and lung cancers (BMLC) and leptomeningeal carcinomatosis (LC). Whole exome sequencing was done in CSF and tumor (n = 3) and was coupled with digital PCR for monitoring CSF and plasma ctDNA (n = 6). Targeted capture massively parallel sequencing of 39 samples from 4 BMBC pts, including samples from CSF, plasma and matched metastatic sites obtained at autopsy was performed using two independent platforms (MSK-IMPACT (341 genes) and BC panel (254 genes most frequently mutated in BC)) comprising 488 unique genes. Sequencing was performed on an Illumina HiSeq2000. Single nucleotide variants were defined using MuTect, and indels using Varscan. Copy number alterations were assessed using Varscan2 and GISTIC. Results: Whole exome sequencing of tumor and CSF DNA revealed a high proportion of tumor-derived cell-free DNA in the CSF of 3 pts (1 GBM, 2 BMBC). We identified actionable somatic mutations (EGFR L858R, IDH1 R132H) and also IDH1 mutations associated with molecular diagnosis of proneural GBM. In CSF and plasma ctDNA of 6 pts with GBM, BMBC, BMLC, we observed that the mutations present in the CSF ctDNA, but not plasma ctDNA, and their mutant allele fractions (MAFs) correlated with brain tumor burden. In 3 pts with suspected LC, we compared the results of cytopathologic analysis and CSF ctDNA obtained from samples used for cytopathologic diagnosis, and observed that CSF ctDNA was more robust and sensitive for the diagnosis of LC. The analysis of synchronous CSF ctDNA, plasma ctDNA and intra- and extra-cranial metastases from 4 autopsied BMBC pts showed that CSF ctDNA recapitulated the somatic genetic alterations present in the intra-cranial lesions. We detected similar MAFs for the truncal mutations (RB1, KMT2D, AHNAK2) in both CSF and plasma DNA of BMBC3, a pt with Li-Fraumeni syndrome and a diagnosis of concurrent BMBC and esthesioneuroblastoma; however, mutations in PIK3CB, PAK7, MSH5 found only in the CNS implant of each disease were only detected in the CSF but not in the plasma DNA. Conclusions: Brain tumor-derived ctDNA is abundantly present in the CSF of brain cancer pts and compared to plasma ctDNA, CSF ctDNA is more representative of the brain lesions. Our results demonstrate that massively parallel sequencing can be performed using CSF DNA, allowing for the non-invasive genomic characterization and monitoring of brain lesions. Citation Format: Leticia De Mattos-Arruda, Regina Mayor, Charlotte K. Y. Ng, Britta Weigelt, Francisco Martinez-Ricarte, Davis Torrejon, Mafalda Oliveira, Carolina Raventos, Alexandra Arias, Elena Guerini-Rocco, Elena Martinez-Saez, Sergio Lois, Oscar Marin, Xavier de la Cruz, Salvatore Piscuoglio, Russell Towers, Ana Vivancos, Vicente Peg, Santiago Ramon y Cajal, Jordi Rodon, Enriqueta Felip, Joan Sahuquillo, Josep Tabernero, Javier Cortes, Jorge S. Reis-Filho, Joan Seoane. Analysis of cell-free tumor DNA in cerebrospinal fluid to characterize and monitor the genetic alterations of brain tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 930. doi:10.1158/1538-7445.AM2015-930

Cell-free circulating tumour DNA (ctDNA) in plasma has been shown to be informative of the genomic alterations present in tumours and has been used to monitor tumour progression and response to treatments. However, patients with brain tumours do not present with or present with low amounts of ctDNA in plasma precluding the genomic characterization of brain cancer through plasma ctDNA. Here we show that ctDNA derived from central nervous system tumours is more abundantly present in the cerebrospinal fluid (CSF) than in plasma. Massively parallel sequencing of CSF ctDNA more comprehensively characterizes the genomic alterations of brain tumours than plasma, allowing the identification of actionable brain tumour somatic mutations. We show that CSF ctDNA levels longitudinally fluctuate in time and follow the changes in brain tumour burden providing biomarkers to monitor brain malignancies. Moreover, CSF ctDNA is shown to facilitate and complement the diagnosis of leptomeningeal carcinomatosis.

Hereditary hemochromatosis (HH) type 3 is an autosomal recessive disorder of iron metabolism characterized by excessive iron deposition in the liver and caused by mutations in the transferrin receptor 2 (TFR2) gene. Here, we describe three new HH type 3 Spanish families with four TFR2 mutations (p.Gly792Arg, c.1606-8A>G, Gln306*, and Gln672*). The missense variation p.Gly792Arg was found in homozygosity in two adult patients of the same family, and in compound heterozygosity in an adult proband that also carries a novel intronic change (c.1606-8A>G). Two new nonsense TFR2 mutations (Gln306* and Gln672*) were detected in a pediatric case. We examine the functional consequences of two TFR2 variants (p.Gly792Arg and c.1606-8A>G) using molecular and computational methods. Cellular protein localization studies using immunofluorescence demonstrated that the plasma membrane localization of p.Gly792Arg TFR2 is impaired. Splicing studies in vitro and in vivo reveal that the c.1606-8A>G mutation leads to the creation of a new acceptor splice site and an aberrant TFR2 mRNA. The reported mutations caused HH type 3 by protein truncation, altering TFR2 membrane localization or by mRNA splicing defect, producing a nonfunctional TFR2 protein and a defective signaling transduction for hepcidin regulation. TFR2 genotyping should be considered in adult but also in pediatric cases with early-onset of iron overload.

Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3) is a genetic disorder caused by mutations in UNC13D gene, coding the granule priming factor Munc13-4 that intervenes in NK and T cell cytotoxic function. Here we report the case of a 17-month-old girl with prolonged symptomatic EBV infectious mononucleosis and clinical symptoms of hemophagocytic syndrome. In vitro functional analysis pointed to a degranulation defect. The genetic analysis of UNC13D gene identified initially a heterozygous mutation (c.753 + 1G > T) in the donor splice-site that resulted in exon 9 skipping (maternal allele). Mutations in other genes were considered, but additional analysis of UNC13D cDNA revealed in the paternal allele a heterozygous transition from G to A (c.2448 − 13G > A) at the 3′ acceptor splice-site in intron 25, generating a new acceptor splice-site that leads to a frameshift and a premature STOP codon. Allele specific amplification of the cDNA confirmed the absence of a functional mRNA from the paternal allele. This case illustrates an atypical compound heterozygous UNC13D mutation affecting the RNA splicing that generates a typical FHL3 phenotype.