Martin Werber

Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana

Basic helix-loop-helix (bHLH) transcription factors represent a family of proteins that contain a bHLH domain, a motif involved in binding DNA. Recently, two groups independently analyzed the BHLH gene family of Arabidopsis thaliana ([Heim et al., 2003][1]; [Toledo-Ortiz et al., 2003][2]). These

Somatic mutation profiles of MSI and MSS colorectal cancer identified by whole exome next generation sequencing and bioinformatics analysis

Background Colorectal cancer (CRC) is with approximately 1 million cases the third most common cancer worldwide. Extensive research is ongoing to decipher the underlying genetic patterns with the hope to improve early cancer diagnosis and treatment. In this direction, the recent progress in next generation sequencing technologies has revolutionized the field of cancer genomics. However, one caveat of these studies remains the large amount of genetic variations identified and their interpretation. Methodology/Principal Findings Here we present the first work on whole exome NGS of primary colon cancers. We performed 454 whole exome pyrosequencing of tumor as well as adjacent not affected normal colonic tissue from microsatellite stable (MSS) and microsatellite instable (MSI) colon cancer patients and identified more than 50,000 small nucleotide variations for each tissue. According to predictions based on MSS and MSI pathomechanisms we identified eight times more somatic non-synonymous variations in MSI cancers than in MSS and we were able to reproduce the result in four additional CRCs. Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions. In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function. Conclusions/Significance We conclude that with deep sequencing of tumor exomes one may be able to predict the microsatellite status of CRC and in addition identify potentially clinically relevant mutations.

Expression of Msgn1 in the presomitic mesoderm is controlled by synergism of WNT signalling and Tbx6

The vertebral column and skeletal muscles of vertebrates are derived from the paraxial mesoderm, which is laid down initially as two stripes of mesenchymal cells alongside the neural tube and subsequently segmented. Previous work has shown that the wingless‐type MMTV integration site family (WNT), fibroblast growth factor‐ and Delta–Notch signalling pathways control presomitic mesoderm (psm) formation and segmentation. Here, we show that the expression of mesogenin 1, a basic helix–loop–helix transcription factor, which is essential for psm maturation, is regulated by synergism between WNT signalling and the T‐box 6 transcription factor, involving a feed‐forward control mechanism. These findings emphasize the crucial role of WNT signalling in the control of psm formation, maturation and segmentation.

Clinical exome sequencing: results from 2819 samples reflecting 1000 families.

We report our results of 1000 diagnostic WES cases based on 2819 sequenced samples from 54 countries with a wide phenotypic spectrum. Clinical information given by the requesting physicians was translated to HPO terms. WES processes were performed according to standardized settings. We identified the underlying pathogenic or likely pathogenic variants in 307 families (30.7%). In further 253 families (25.3%) a variant of unknown significance, possibly explaining the clinical symptoms of the index patient was identified. WES enabled timely diagnosing of genetic diseases, validation of causality of specific genetic disorders of PTPN23, KCTD3, SCN3A, PPOX, FRMPD4, and SCN1B, and setting dual diagnoses by detecting two causative variants in distinct genes in the same patient. We observed a better diagnostic yield in consanguineous families, in severe and in syndromic phenotypes. Our results suggest that WES has a better yield in patients that present with several symptoms, rather than an isolated abnormality. We also validate the clinical benefit of WES as an effective diagnostic tool, particularly in nonspecific or heterogeneous phenotypes. We recommend WES as a first-line diagnostic in all cases without a clear differential diagnosis, to facilitate personal medical care.

The Saccharomyces cerevisiae W303-K6001 cross-platform genome sequence: insights into ancestry and physiology of a laboratory mutt

Saccharomyces cerevisiae strain W303 is a widely used model organism. However, little is known about its genetic origins, as it was created in the 1970s from crossing yeast strains of uncertain genealogy. To obtain insights into its ancestry and physiology, we sequenced the genome of its variant W303-K6001, a yeast model of ageing research. The combination of two next-generation sequencing (NGS) technologies (Illumina and Roche/454 sequencing) yielded an 11.8 Mb genome assembly at an N50 contig length of 262 kb. Although sequencing was substantially more precise and sensitive than whole-genome tiling arrays, both NGS platforms produced a number of false positives. At a 378× average coverage, only 74 per cent of called differences to the S288c reference genome were confirmed by both techniques. The consensus W303-K6001 genome differs in 8133 positions from S288c, predicting altered amino acid sequence in 799 proteins, including factors of ageing and stress resistance. The W303-K6001 (85.4%) genome is virtually identical (less than equal to 0.5 variations per kb) to S288c, and thus originates in the same ancestor. Non-S288c regions distribute unequally over the genome, with chromosome XVI the most (99.6%) and chromosome XI the least (54.5%) S288c-like. Several of these clusters are shared with Σ1278B, another widely used S288c-related model, indicating that these strains share a second ancestor. Thus, the W303-K6001 genome pictures details of complex genetic relationships between the model strains that date back to the early days of experimental yeast genetics. Moreover, this study underlines the necessity of combining multiple NGS and genome-assembling techniques for achieving accurate variant calling in genomic studies.

High-throughput sequencing of microdissected chromosomal regions

The linkage of disease gene mapping with DNA sequencing is an essential strategy for defining the genetic basis of a disease. New massively parallel sequencing procedures will greatly facilitate this process, although enrichment for the target region before sequencing remains necessary. For this step, various DNA capture approaches have been described that rely on sequence-defined probe sets. To avoid making assumptions on the sequences present in the targeted region, we accessed specific cytogenetic regions in preparation for next-generation sequencing. We directly microdissected the target region in metaphase chromosomes, amplified it by degenerate oligonucleotide-primed PCR, and obtained sufficient material of high quality for high-throughput sequencing. Sequence reads could be obtained from as few as six chromosomal fragments. The power of cytogenetic enrichment followed by next-generation sequencing is that it does not depend on earlier knowledge of sequences in the region being studied. Accordingly, this method is uniquely suited for situations in which the sequence of a reference region of the genome is not available, including population-specific or tumor rearrangements, as well as previously unsequenced genomic regions such as centromeres.

Retention of gene products in syncytial spermatids promotes non-Mendelian inheritance as revealed by the t complex responder

The t complex responder (Tcr) encoded by the mouse t haplotype is able to cause phenotypic differences between t and + sperm derived from t/+ males, leading to non-Mendelian inheritance. This capability of Tcr contradicts the concept of phenotypic equivalence proposed for sperm cells, which develop in a syncytium and actively share gene products. By analyzing a Tcr minigene in hemizygous transgenic mice, we show that Tcr gene products are post-meiotically expressed and are retained in the haploid sperm cells. The wild-type allele of Tcr, sperm motility kinase-1 (Smok1), behaves in the same manner, suggesting that Tcr/Smok reveal a common mechanism prone to evolve non-Mendelian inheritance in mammals.

Using the canary genome to decipher the evolution of hormone-sensitive gene regulation in seasonal singing birds

BackgroundWhile the song of all songbirds is controlled by the same neural circuit, the hormone dependence of singing behavior varies greatly between species. For this reason, songbirds are ideal organisms to study ultimate and proximate mechanisms of hormone-dependent behavior and neuronal plasticity.ResultsWe present the high quality assembly and annotation of a female 1.2-Gbp canary genome. Whole genome alignments between the canary and 13 genomes throughout the bird taxa show a much-conserved synteny, whereas at the single-base resolution there are considerable species differences. These differences impact small sequence motifs like transcription factor binding sites such as estrogen response elements and androgen response elements. To relate these species-specific response elements to the hormone-sensitivity of the canary singing behavior, we identify seasonal testosterone-sensitive transcriptomes of major song-related brain regions, HVC and RA, and find the seasonal gene networks related to neuronal differentiation only in the HVC. Testosterone-sensitive up-regulated gene networks of HVC of singing males concerned neuronal differentiation. Among the testosterone-regulated genes of canary HVC, 20% lack estrogen response elements and 4 to 8% lack androgen response elements in orthologous promoters in the zebra finch.ConclusionsThe canary genome sequence and complementary expression analysis reveal intra-regional evolutionary changes in a multi-regional neural circuit controlling seasonal singing behavior and identify gene evolution related to the hormone-sensitivity of this seasonal singing behavior. Such genes that are testosterone- and estrogen-sensitive specifically in the canary and that are involved in rewiring of neurons might be crucial for seasonal re-differentiation of HVC underlying seasonal song patterning.

The tissue-specific transcriptomic landscape of the mid-gestational mouse embryo

Differential gene expression is a prerequisite for the formation of multiple cell types from the fertilized egg during embryogenesis. Understanding the gene regulatory networks controlling cellular differentiation requires the identification of crucial differentially expressed control genes and, ideally, the determination of the complete transcriptomes of each individual cell type. Here, we have analyzed the transcriptomes of six major tissues dissected from mid-gestational (TS12) mouse embryos. Approximately one billion reads derived by RNA-seq analysis provided extended transcript lengths, novel first exons and alternative transcripts of known genes. We have identified 1375 genes showing tissue-specific expression, providing gene signatures for each of the six tissues. In addition, we have identified 1403 novel putative long noncoding RNA gene loci, 439 of which show differential expression. Our analysis provides the first complete transcriptome data for the mouse embryo. It offers a rich data source for the analysis of individual genes and gene regulatory networks controlling mid-gestational development.

Validation of a semiconductor next‐generation sequencing assay for the clinical genetic screening of CFTR

Genetic testing for cystic fibrosis and CFTR‐related disorders mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the CFTR gene. We have explored a more efficient genetic screening strategy based on next‐generation sequencing (NGS) of the CFTR gene. We validated this approach in a cohort of 177 patients with previously known CFTR mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq™ CFTR panel. The DNA libraries were pooled, barcoded, and sequenced using an Ion Torrent PGM sequencer. The combination of different robust bioinformatics tools allowed us to detect previously known pathogenic mutations and polymorphisms in the 177 samples, without detecting spurious pathogenic calls. In summary, the assay achieves a sensitivity of 94.45% (95% CI: 92% to 96.9%), with a specificity of detecting nonvariant sites from the CFTR reference sequence of 100% (95% CI: 100% to 100%), a positive predictive value of 100% (95% CI: 100% to 100%), and a negative predictive value of 99.99% (95% CI: 99.99% to 100%). In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases. Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly‐thymidine‐guanine and poly‐thymidine (poly‐TG‐T) tracts were also detected. Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.