A full-body transcriptome and proteome resource for the European common carp.

BACKGROUND: The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies. RESULTS: Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data. CONCLUSIONS: The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.

Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting.

This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.

Collembolan Transcriptomes Highlight Molecular Evolution of Hexapods and Provide Clues on the Adaptation to Terrestrial Life.

BACKGROUND: Collembola (springtails) represent a soil-living lineage of hexapods in between insects and crustaceans. Consequently, their genomes may hold key information on the early processes leading to evolution of Hexapoda from a crustacean ancestor. METHOD: We assembled and annotated transcriptomes of the Collembola Folsomia candida and Orchesella cincta, and performed comparative analysis with protein-coding gene sequences of three crustaceans and three insects to identify adaptive signatures associated with the evolution of hexapods within the pancrustacean clade. RESULTS: Assembly of the springtail transcriptomes resulted in 37,730 transcripts with predicted open reading frames for F. candida and 32,154 for O. cincta, of which 34.2% were functionally annotated for F. candida and 38.4% for O. cincta. Subsequently, we predicted orthologous clusters among eight species and applied the branch-site test to detect episodic positive selection in the Hexapoda and Collembola lineages. A subset of 250 genes showed significant positive selection along the Hexapoda branch and 57 in the Collembola lineage. Gene Ontology categories enriched in these genes include metabolism, stress response (i.e. DNA repair, immune response), ion transport, ATP metabolism, regulation and development-related processes (i.e. eye development, neurological development). CONCLUSIONS: We suggest that the identified gene families represent processes that have played a key role in the divergence of hexapods within the pancrustacean clade that eventually evolved into the most species-rich group of all animals, the hexapods. Furthermore, some adaptive signatures in collembolans may provide valuable clues to understand evolution of hexapods on land.

Next generation sequencing technology: Advances and applications.

Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing reaction has profoundly increased the total number of produced sequence reads per run. Current sequencing platforms allow for a previously unprecedented view into complex mixtures of RNA and DNA samples. NGS is currently evolving into a molecular microscope finding its way into virtually every fields of biomedical research. In this chapter we review the technical background of the different commercially available NGS platforms with respect to template generation and the sequencing reaction and take a small step towards what the upcoming NGS technologies will bring. We close with an overview of different implementations of NGS into biomedical research. This article is part of a Special Issue entitled: From Genome to Function.

Molecular diagnostics of the HBB gene in an Omani cohort using bench-top DNA Ion Torrent PGM technology.

Hemoglobinopathies, such as sickle cell disease (SCD) and beta-thalassemia major (TM), are severe diseases and the most common autosomal recessive condition worldwide and in particular in Oman. Early screening and diagnosis of carriers are the key for primary prevention. Once a country-wide population screening program is mandated by law, a sequencing technology that can rapidly confirm or identify disease-causing mutations for a large number of patients in a short period of time will be necessary. While Sanger sequencing is the standard protocol for molecular diagnosis, next generation sequencing starts to become available to reference laboratories. Using the Ion Torrent PGM sequencer, we have analyzed a cohort of 297 unrelated Omani cases and reliably identified mutations in the beta-globin (HBB) gene. Our model study has shown that Ion Torrent PGM can rapidly sequence such a small gene in a large number of samples using a barcoded uni-directional or bi-directional sequence methodology, reducing cost, workload and providing accurate diagnosis. Based on our results we believe that the Ion Torrent PGM sequencing platform, able to analyze hundreds of patients simultaneously for a single disease gene can be a valid molecular screening alternative to ABI sequencing in the diagnosis of hemoglobinopathies and other genetic disorders in the near future.

The InSiGHT database: utilizing 100 years of insights into Lynch syndrome.

This article provides a historical overview of the online database ( www.insight-group.org/mutations ) maintained by the International Society for Gastrointestinal Hereditary Tumours. The focus is on the mismatch repair genes which are mutated in Lynch Syndrome. APC, MUTYH and other genes are also an important part of the database, but are not covered here. Over time, as the understanding of the genetics of Lynch Syndrome increased, databases were created to centralise and share the variants which were being detected in ever greater numbers. These databases were eventually merged into the InSiGHT database, a comprehensive repository of gene variant and disease phenotype information, serving as a starting point for important endeavours including variant interpretation, research, diagnostics and enhanced global collection. Pivotal to its success has been the collaborative spirit in which it has been developed, its association with the Human Variome Project, the appointment of a full time curator and its governance stemming from the well established organizational structure of InSiGHT.

Serum matrix metalloproteinase-9 (MMP-9) as a biomarker for monitoring disease progression in Duchenne muscular dystrophy (DMD).

To identify serum biomarkers that allow monitoring of disease progression and treatment effects in Duchenne muscular dystrophy (DMD) patients, levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1) and osteopontin (OPN) were determined in 63 DMD patients on corticosteroid therapy. These proteins were selected for their role in the pathogenesis of muscular dystrophy. Levels of MMP-9 and TIMP-1 were significantly higher in sera of DMD patients compared to healthy controls, whereas the OPN levels showed no significant difference. MMP-9 levels were also observed to be significantly higher in older, nonambulant patients, compared to ambulant patients. Longitudinal data from a smaller cohort of DMD patients followed up for over 4years showed that MMP-9, but not TIMP-1 increased significantly with age. Hence, MMP-9 is a potential DMD biomarker for disease progression. Future studies have to confirm whether serum MMP-9 levels can be used to monitor therapeutic response.

Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients.

Theoretically, 13% of patients with Duchenne muscular dystrophy may benefit from antisense-mediated skipping of exon 51 to restore the reading frame, which results in the production of a shortened dystrophin protein. We give a detailed description with longitudinal follow up of three patients with Becker muscular dystrophy with in-frame deletions in the DMD gene encompassing exon 51. Their internally deleted, but essentially functional, dystrophins are identical to those that are expected as end products in DMD patients treated with the exon 51 skipping therapy. The mild phenotype encourages further development of exon 51 skipping therapy.

Relative power and sample size analysis on gene expression profiling data.

BACKGROUND: With the increasing number of expression profiling technologies, researchers today are confronted with choosing the technology that has sufficient power with minimal sample size, in order to reduce cost and time. These depend on data variability, partly determined by sample type, preparation and processing. Objective measures that help experimental design, given own pilot data, are thus fundamental. RESULTS: Relative power and sample size analysis were performed on two distinct data sets. The first set consisted of Affymetrix array data derived from a nutrigenomics experiment in which weak, intermediate and strong PPARalpha agonists were administered to wild-type and PPARalpha-null mice. Our analysis confirms the hierarchy of PPARalpha-activating compounds previously reported and the general idea that larger effect sizes positively contribute to the average power of the experiment. A simulation experiment was performed that mimicked the effect sizes seen in the first data set. The relative power was predicted but the estimates were slightly conservative. The second, more challenging, data set describes a microarray platform comparison study using hippocampal deltaC-doublecortin-like kinase transgenic mice that were compared to wild-type mice, which was combined with results from Solexa/Illumina deep sequencing runs. As expected, the choice of technology greatly influences the performance of the experiment. Solexa/Illumina deep sequencing has the highest overall power followed by the microarray platforms Agilent and Affymetrix. Interestingly, Solexa/Illumina deep sequencing displays comparable power across all intensity ranges, in contrast with microarray platforms that have decreased power in the low intensity range due to background noise. This means that deep sequencing technology is especially more powerful in detecting differences in the low intensity range, compared to microarray platforms. CONCLUSION: Power and sample size analysis based on pilot data give valuable information on the performance of the experiment and can thereby guide further decisions on experimental design. Solexa/Illumina deep sequencing is the technology of choice if interest lies in genes expressed in the low-intensity range. Researchers can get guidance on experimental design using our approach on their own pilot data implemented as a BioConductor package, SSPA http://bioconductor.org/packages/release/bioc/html/SSPA.html.

Recurrence risk due to germ line mosaicism: Duchenne and Becker muscular dystrophy.

The presence of multiple affected offspring from apparently non-carrier parents is caused by germ line mosaicism. Although germ line mosaicism has been reported for many diseases, figures for recurrence risks are known for only a few of them. In X-linked Duchenne and Becker muscular dystrophies (DMD/BMD), the recurrence risk for non-carrier females due to germ line mosaicism has been estimated to be between 14% and 20% (95% confidence interval 3-30) if the risk haplotype is transmitted. In this study, we have analyzed 318 DMD/BMD cases in which the detected mutation was de novo with the aim of obtaining a better estimate of the 'true' number of germ line mosaics and a more precise recurrence risk. This knowledge is essential for genetic counseling. Our data indicate a recurrence risk of 8.6% (4.8-12.2) if the risk haplotype is transmitted, but there is a remarkable difference between proximal (15.6%) (4.1-27.0) and distal (6.4%) (2.1-10.6) deletions. Overall, most mutations originated in the female. Deletions occur more often on the X chromosome of the maternal grandmother, whereas point mutations occur on the X chromosome of the maternal grandfather. In unhaplotyped de novo DMD/BMD families, the risk of recurrence of the mutation is 4.3%.

Somatic mutation databases as tools for molecular epidemiology and molecular pathology of cancer: proposed guidelines for improving data collection, distribution, and integration.

There are currently less than 40 locus-specific databases (LSDBs) and one large general database that curate data on somatic mutations in human cancer genes. These databases have different scope and use different annotation standards and database systems, resulting in duplicated efforts in data curation, and making it difficult for users to find clear and consistent information. As data related to somatic mutations are generated at an increasing pace it is urgent to create a framework for improving the collecting of this information and making it more accessible to clinicians, scientists, and epidemiologists to facilitate research on biomarkers. Here we propose a data flow for improving the connectivity between existing databases and we provide practical guidelines for data reporting, database contents, and annotation standards. These proposals are based on common standards recommended by the Human Genome Variation Society (HGVS) with additions related to specific requirements of somatic mutations in cancer. Indeed, somatic mutations may be used in molecular pathology and clinical studies to characterize tumor types, help treatment choice, predict response to treatment and patient outcome, or in epidemiological studies as markers for tumor etiology or exposure assessment. Thus, specific annotations are required to cover these diverse research topics. This initiative is meant to promote collaboration and discussion on these issues and the development of adequate resources that would avoid the loss of extremely valuable information generated by years of basic and clinical research.

Recommendations for locus-specific databases and their curation.

Expert curation and complete collection of mutations in genes that affect human health is essential for proper genetic healthcare and research. Expert curation is given by the curators of gene-specific mutation databases or locus-specific databases (LSDBs). While there are over 700 such databases, they vary in their content, completeness, time available for curation, and the expertise of the curator. Curation and LSDBs have been discussed, written about, and protocols have been provided for over 10 years, but there have been no formal recommendations for the ideal form of these entities. This work initiates a discussion on this topic to assist future efforts in human genetics. Further discussion is welcome.

Methods to detect CNVs in the human genome.

The detection of quantitative changes in genomic DNA, i.e. deletions and duplications or Copy Number Variants (CNVs), has recently gained considerable interest. First, detailed analysis of the human genome showed a surprising amount of CNVs, involving thousands of genes. Second, it was realised that the detection of CNVs as a cause of genetic disease was often neglected, but should be an essential part of a complete screening strategy. In both cases new efficient CNV screening methods, covering the entire range from specific loci to genome-wide, were behind these developments. This paper will briefly review the methods that are available to detect CNVs, discuss their strong and weak points, show some new developments and look ahead. Methods covered include microscopy, fluorescence in situ hybridization (including fiber-FISH), Southern blotting, PCR-based methods (including MLPA), array technology and massive parallel sequencing. In addition, we will show some new developments, including a 1400-plex CNV bead assay, fast-MLPA (from DNA to result in approximately 6 h) and a simple Melting Curve Analysis assay to confirm potential CNVs. Using the 1400-plex CNV bead assay, targeting selected chromosomal regions only, we detected confirmed rearrangements in 9% of 320 mental retardation patients studied.

Variation of CNV distribution in five different ethnic populations.

Recent studies have revealed a new type of variation in the human genome encompassing relatively large genomic segments ( approximately 100 kb-2.5 Mb), commonly referred to as copy number variation (CNV). The full nature and extent of CNV and its frequency in different ethnic populations is still largely unknown. In this study we surveyed a set of 12 CNVs previously detected by array-CGH. More than 300 individuals from five different ethnic populations, including three distinct European, one Asian and one African population, were tested for the occurrence of CNV using multiplex ligation-dependent probe amplification (MLPA). Seven of these loci indeed showed CNV, i.e., showed copy numbers that deviated from the population median. More precise estimations of the actual genomic copy numbers for (part of) the NSF gene locus, revealed copy numbers ranging from two to at least seven. Additionally, significant inter-population differences in the distribution of these copy numbers were observed. These data suggest that insight into absolute DNA copy numbers for loci exhibiting CNV is required to determine their potential contribution to normal phenotypic variation and, in addition, disease susceptibility.

A structured simple form for ordering genetic tests is needed to ensure coupling of clinical detail (phenotype) with DNA variants (genotype) to ensure utility in publication and databases.

Researchers and clinicians ideally need instant access to all the variation in their gene/locus of interest to efficiently conduct their research and genetic healthcare to the highest standards. Currently much key data resides in the laboratory books or patient records around the world, as there are many impediments to submitting this data. It would be ideal therefore if a semiautomated pathway was available, with a minimum of effort, to make the deidentified data publicly available for others to use. The Human Variome Project (HVP) meeting listed 96 recommendations to work toward this situation. This article is planned to initiate a strategy to enhance the collection of phenotype and genotype data from the clinician/diagnostic laboratory nexus. Thus, the aim is to develop universally applicable forms that people can use when investigating patients for each inherited disease, to assist in satisfying many of the recommendations of the HVP Meeting [Cotton et al., 2007]. We call for comment and collaboration in this article.

Copy number variation in the genome; the human DMD gene as an example.

Recent developments have yielded new technologies that have greatly simplified the detection of deletions and duplications, i.e., copy number variants (CNVs). These technologies can be used to screen for CNVs in and around specific genomic regions, as well as genome-wide. Several genome-wide studies have demonstrated that CNV in the human genome is widespread and may include millions of nucleotides. One of the questions that emerge is which sequences, structures and/or processes are involved in their generation. Using as an example the human DMD gene, mutations in which cause Duchenne and Becker muscular dystrophy, we review the current data, determine the deletion and duplication profile across the gene and summarize the information that has been collected regarding their origin. In addition we discuss the methods most frequently used for their detection, in particular MAPH and MLPA.

Duplications in the DMD gene.

The detection of duplications in Duchenne (DMD)/Becker Muscular Dystrophy (BMD) has long been a neglected issue. However, recent technological advancements have significantly simplified screening for such rearrangements. We report here the detection and analysis of 118 duplications in the DMD gene of DMD/BMD patients. In an unselected patient series the duplication frequency was 7%. In patients already screened for deletions and point mutations, duplications were detected in 87% of cases. There were four complex, noncontiguous rearrangements, with two also involving a partial triplication. In one of the few cases where RNA was analyzed, a seemingly contiguous duplication turned out to be a duplication/deletion case generating a transcript with an unexpected single-exon deletion and an initially undetected duplication. These findings indicate that for clinical diagnosis, duplications should be treated with special care, and without further analysis the reading frame rule should not be applied. As with deletions, duplications occur nonrandomly but with a dramatically different distribution. Duplication frequency is highest near the 5' end of the gene, with a duplication of exon 2 being the single most common duplication identified. Analysis of the extent of 11 exon 2 duplications revealed two intron 2 recombination hotspots. Sequencing four of the breakpoints showed that they did not arise from unequal sister chromatid exchange, but more likely from synthesis-dependent nonhomologous end joining. There appear to be fundamental differences therefore in the origin of deletions and duplications in the DMD gene.

Common pathological mechanisms in mouse models for muscular dystrophies.

Duchenne/Becker and limb-girdle muscular dystrophies share clinical symptoms like muscle weakness and wasting but differ in clinical presentation and severity. To get a closer view on the differentiating molecular events responsible for the muscular dystrophies, we have carried out a comparative gene expression profiling of hindlimb muscles of the following mouse models: dystrophin-deficient (mdx, mdx(3cv)), sarcoglycan-deficient (Sgca null, Sgcb null, Sgcg null, Sgcd null), dysferlin-deficient (Dysf null, SJL(Dysf)), sarcospan-deficient (Sspn null), and wild-type (C57Bl/6, C57Bl/10) mice. The expression profiles clearly discriminated between severely affected (dystrophinopathies and sarcoglycanopathies) and mildly or nonaffected models (dysferlinopathies, sarcospan-deficiency, wild-type). Dystrophin-deficient and sarcoglycan-deficient profiles were remarkably similar, sharing inflammatory and structural remodeling processes. These processes were also ongoing in dysferlin-deficient animals, albeit at lower levels, in agreement with the later age of onset of this muscular dystrophy. The inflammatory proteins Spp1 and S100a9 were up-regulated in all models, including sarcospan-deficient mice, which points, for the first time, at a subtle phenotype for Sspn null mice. In conclusion, we identified biomarker genes for which expression correlates with the severity of the disease, which can be used for monitoring disease progression. This comparative study is an integrating step toward the development of an expression profiling-based diagnostic approach for muscular dystrophies in humans.

Muscle regeneration in dystrophin-deficient mdx mice studied by gene expression profiling.

BACKGROUND: Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is lethal. In contrast, dystrophin-deficient mdx mice recover due to effective regeneration of affected muscle tissue. To characterize the molecular processes associated with regeneration, we compared gene expression levels in hindlimb muscle tissue of mdx and control mice at 9 timepoints, ranging from 1-20 weeks of age. RESULTS: Out of 7776 genes, 1735 were differentially expressed between mdx and control muscle at at least one timepoint (p < 0.05 after Bonferroni correction). We found that genes coding for components of the dystrophin-associated glycoprotein complex are generally downregulated in the mdx mouse. Based on functional characteristics such as membrane localization, signal transduction, and transcriptional activation, 166 differentially expressed genes with possible functions in regeneration were analyzed in more detail. The majority of these genes peak at the age of 8 weeks, where the regeneration activity is maximal. The following pathways are activated, as shown by upregulation of multiple members per signalling pathway: the Notch-Delta pathway that plays a role in the activation of satellite cells, and the Bmp15 and Neuregulin 3 signalling pathways that may regulate proliferation and differentiation of satellite cells. In DMD patients, only few of the identified regeneration-associated genes were found activated, indicating less efficient regeneration processes in humans. CONCLUSION: Based on the observed expression profiles, we describe a model for muscle regeneration in mdx mice, which may provide new leads for development of DMD therapies based on the improvement of muscle regeneration efficacy.

Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification.

BACKGROUND: Approximately 80% of the alpha- and 10% of the beta-thalassaemias are caused by genomic deletions involving the alpha- and beta-globin gene clusters on chromosomes 16p13.3 and 11p15.5, respectively. Gap-PCR, Southern blot analysis, and fluorescent in situ hybridisation are commonly used to identify these deletions; however, many deletions go undetected using conventional techniques. METHODS: Patient samples for which no abnormalities had been found using conventional DNA techniques were analysed by a three colour multiplex ligation-dependent probe amplification assay. Two sets of 35 and 50 probes, covering a region of 700 kb of the alpha- and 500 kb of the beta-globin gene cluster, respectively, were designed to detect rearrangements in the alpha- and beta-globin gene clusters. RESULTS: In 19 out of 38 patient samples, we found 11 different alpha-thalassaemia deletions, six of which were not previously described. Two novel deletions leaving the alpha-globin gene cluster intact were found to cause a complete downregulation of the downstream alpha-genes. Similarly, 31 out of 51 patient samples were found to carry 10 different deletions involving the beta-globin gene cluster, three of which were not previously described. One involves the deletion of the locus control region leaving the beta-globin gene cluster intact. CONCLUSIONS: These deletions, which are not easily detected by conventional techniques, may have clinical implications during pregnancy ranging from mild to life threatening microcytic haemolytic anaemia in neonates. The approach as described here is a rapid and sensitive method for high resolution analysis of the globin gene clusters and for any region of the genome.