Browsing by Author "Becker, Christian"

We received early access to the newest releases of exome sequencing products, namely Agilent SureSelect v6 (Agilent, Santa Clara, CA, USA) and NimbleGen MedExome (Roche NimbleGen, Basel, Switzerland), and we conducted whole exome sequencing (WES) of several DNA samples with each of these products in order to assess their performance. Here, we provide a detailed evaluation of the original, normalized (with respect to the different target sizes), and trimmed data sets and compare them in terms of the amount of duplicates, the reads on target, and the enrichment evenness. In addition to these general statistics, we performed a detailed analysis of the frequently mutated and newly described genes found in 'The Deciphering Developmental Disorders Study' published very recently (Fitzgerald, T.W., Gerety, S.S., Jones, W.D., van Kogelenberg, M., King, D.A., McRae, J., Morley, K.I., Parthiban, V., Al-Turki, S., Ambridge, K., et al. (2015). Large-scale discovery of novel genetic causes of developmental disorders. Nature 519, 223-228.). In our comparison, the Agilent v6 exome performs better than the NimbleGen's MedExome both in terms of efficiency and evenness of coverage distribution. With its larger target size, it is also more comprehensive, and therefore the better choice in research projects that aim to identify novel disease-associated genes. In contrast, if the exomes are mainly used in a diagnostic setting, we see advantages for the new NimbleGen MedExome. We find a superior coverage here in those genes of high clinical relevance that likely allows for a better detection of relevant, disease-causing mutations.

Crisponi syndrome is a severe autosomal recessive condition that is phenotypically characterized by abnormal, paroxysmal muscular contractions resembling neonatal tetanus, large face, broad nose, anteverted nares, camptodactyly, hyperthermia, and sudden death in most cases. We performed homozygosity mapping in five Sardinian and three Turkish families with Crisponi syndrome, using high-density single-nucleotide polymorphism arrays, and identified a critical region on chromosome 19p12-13.1. The most prominent candidate gene was CRLF1, recently found to be involved in the pathogenesis of cold-induced sweating syndrome type 1 (CISS1). CISS1 belongs to a group of conditions with overlapping phenotypes, also including cold-induced sweating syndrome type 2 and Stuve-Wiedemann syndrome. All these syndromes are caused by mutations of genes of the ciliary neurotrophic factor ( CNTF) - receptor pathway. Here, we describe the identification of four different CRLF1 mutations in eight different Crisponi-affected families, including a missense mutation, a single-nucleotide insertion, and a nonsense and an insertion/deletion (indel) mutation, all segregating with the disease trait in the families. Comparison of the mutation spectra of Crisponi syndrome and CISS1 suggests that neither the type nor the location of the CRLF1 mutations points to a phenotype/genotype correlation that would account for the most severe phenotype in Crisponi syndrome. Other, still-unknown molecular factors may be responsible for the variable phenotypic expression of the CRLF1 mutations. We suggest that the syndromes can comprise a family of "CNTF-receptor - related disorders," of which Crisponi syndrome would be the newest member and allelic to CISS1.

The ability of animals to sense and differentiate among thousands of odorants relies on a large set of olfactory receptors (OR) and a multitude of accessory proteins within the olfactory epithelium (OE). ORs and related signaling mechanisms have been the subject of intensive studies over the past years, but our knowledge regarding olfactory processing remains limited. The recent development of next generation sequencing (NGS) techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS)-sorted olfactory receptor neurons (ORNs) obtained from transgenic OMP-GFP mice. The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome. In OE samples, nearly all OR and trace amine-associated receptor (TAAR) genes involved in the perception of volatile amines were detectably expressed. Other genes known to participate in olfactory signaling pathways were among the 200 genes with the highest expression levels in the OE. To identify OE-specific genes, we compared olfactory neuron expression profiles with RNA-Seq transcriptome data from different murine tissues. By analyzing different transcript classes, we detected the expression of non-olfactory GPCRs in ORNs and established an expression ranking for GPCRs detected in the OE. We also identified other previously undescribed membrane proteins as potential new players in olfaction. The quantitative and comprehensive transcriptome data provide a virtually complete catalogue of genes expressed in the OE and present a useful tool to uncover candidate genes involved in, for example, olfactory signaling, OR trafficking and recycling, and proliferation.

Purpose: To identify novel mechanisms of resistance to third-generation EGFR inhibitors in patients with lung adenocarcinoma that progressed under therapy with either AZD9291 or rociletinib (CO-1686). Experimental Design: We analyzed tumor biopsies from seven patients obtained before, during, and/or after treatment with AZD9291 or rociletinib (CO-1686). Targeted sequencing and FISH analyses were performed, and the relevance of candidate genes was functionally assessed in in vitro models. Results: We found recurrent amplification of either MET or ERBB2 in tumors that were resistant or developed resistance to third-generation EGFR inhibitors and show that ERBB2 and MET activation can confer resistance to these compounds. Furthermore, we identified a KRAS(G12S) mutation in a patient with acquired resistance to AZD9291 as a potential driver of acquired resistance. Finally, we show that dual inhibition of EGFR/MEK might be a viable strategy to overcome resistance in EGFR-mutant cells expressing mutant KRAS. Conclusions: Our data suggest that heterogeneous mechanisms of resistance can drive primary and acquired resistance to third-generation EGFR inhibitors and provide a rationale for potential combination strategies. (C) 2016 AACR.

Abstract Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome‐wide in two primary cell lines. We integrated ChIP‐seq and Hi‐C with graph theory to uncover clustering of HMGB1‐marked topological domains that harbor genes involved in paracrine senescence. Using simplified Cross‐Linking and Immuno‐Precipitation and functional tests, we show that HMGB1 is also a bona fide RNA‐binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate availability of SASP‐relevant transcripts. Our findings reveal a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell‐autonomous and paracrine senescence.

Purpose: The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland. Methods: In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies. Results: This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3'-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland. Conclusions: RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families.

Cenani-Lenz syndrome (CLS) is an autosomal-recessive congenital disorder affecting distal limb development. It is characterized mainly by syndactyly and/or oligodactyly and is now shown to be commonly associated with kidney anomalies. We used a homozygosity-mapping approach to map the CLS1 locus to chromosome 11p11.2-q13.1. By sequencing candidate genes, we identified recessive LRP4 mutations in 12 families with CLS. LRP4 belongs to the low-density lipoprotein (LDL) receptor-related proteins (LRPs), which are essential for various developmental processes. LRP4 is known to antagonize LRP6-mediated activation of canonical Wnt signaling, a function that is lost by the identified mutations. Our findings increase the spectrum of congenital anomalies associated with abnormal lipoprotein receptor-dependent signaling.

Pontocerebellar hypoplasias (PCH) represent a group of neurodegenerative autosomal recessive disorders with prenatal onset, atrophy or hypoplasia of the cerebellum, hypoplasia of the ventral pons, microcephaly, variable neocortical atrophy and severe mental and motor impairments. In two subtypes, PCH2 and PCH4, we identified mutations in three of the four different subunits of the tRNA-splicing endonuclease complex. Our findings point to RNA processing as a new basic cellular impairment in neurological disorders.