Jeroen Van Houdt

Heterozygous missense mutations in SMARCA2 cause Nicolaides-Baraitser syndrome

Nicolaides-Baraitser syndrome (NBS) is characterized by sparse hair, distinctive facial morphology, distal-limb anomalies and intellectual disability. We sequenced the exomes of ten individuals with NBS and identified heterozygous variants in SMARCA2 in eight of them. Extended molecular screening identified nonsynonymous SMARCA2 mutations in 36 of 44 individuals with NBS; these mutations were confirmed to be de novo when parental samples were available. SMARCA2 encodes the core catalytic unit of the SWI/SNF ATP-dependent chromatin remodeling complex that is involved in the regulation of gene transcription. The mutations cluster within sequences that encode ultra-conserved motifs in the catalytic ATPase region of the protein. These alterations likely do not impair SWI/SNF complex assembly but may be associated with disrupted ATPase activity. The identification of SMARCA2 mutations in humans provides insight into the function of the Snf2 helicase family.

Divergent selection as revealed by P(ST) and QTL-based F(ST) in three-spined stickleback (Gasterosteus aculeatus) populations along a coastal-inland gradient

Three measures of divergence, estimated at nine putatively neutral microsatellite markers, 14 quantitative traits, and seven quantitative trait loci (QTL) were compared in eight populations of the three‐spined stickleback (Gasterosteus aculeatus L.) living in the Scheldt river basin (Belgium). Lowland estuarine and polder populations were polymorphic for the number of lateral plates, whereas upland freshwater populations were low‐plated. The number of short gill rakers and the length of dorsal and pelvic spines gradually declined along a coastal‐inland gradient. Plate number, short gill rakers and spine length showed moderate to strong signals of divergent selection between lowland and upland populations in comparison between PST (a phenotypic alternative for QST) and neutral FST. However, such comparisons rely on the unrealistic assumption that phenotypic variance equals additive genetic variance, and that nonadditive genetic effects and environmental effects can be minimized. In order to verify this assumption and to confirm the phenotypic signals of divergence, we tested for divergent selection at the underlying QTL. For plate number, strong genetic evidence for divergent selection between lowland and upland populations was obtained based on an intron marker of the Eda gene, of which the genotype was highly congruent with plate morph. Genetic evidence for divergent selection on short gill rakers was limited to some population pairs where FST at only one of two QTL was detected as an outlier, although FST at both loci correlated significantly with PST. No genetic confirmation was obtained for divergent selection on dorsal spine length, as no outlier FSTs were detected at dorsal spine QTL, and no significant correlations with PST were observed.

SNP Discovery Using Next Generation Transcriptomic Sequencing in Atlantic Herring (Clupea harengus)

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.

De novo transcriptome assembly and SNP discovery in the wing polymorphic salt marsh beetle Pogonus chalceus (Coleoptera, Carabidae).

Background The salt marsh beetle Pogonus chalceus represents a unique opportunity to understand and study the origin and evolution of dispersal polymorphisms as remarkable inter-population divergence in dispersal related traits (e.g. wing development, body size and metabolism) has been shown to persist in face of strong homogenizing gene flow. Sequencing and assembling the transcriptome of P. chalceus is a first step in developing large scale genetic information that will allow us to further study the recurrent phenotypic evolution in dispersal traits in these natural populations. Methodology/Results We used the Illumina HiSeq2000 to sequence 37 Gbases of the transcriptome and performed de novo transcriptome assembly with the Trinity short read assembler. This resulted in 65,766 contigs, clustering into 39,393 unique transcripts (unigenes). A subset of 12,987 show similarity (BLAST) to known proteins in the NCBI database and 7,589 are assigned Gene Ontology (GO). Using homology searches we identified all reported genes involved in wing development, juvenile- and ecdysteroid hormone pathways in Tribolium castaneum. About half (56.7%) of the unique assembled genes are shared among three life stages (third-instar larva, pupa, and imago). We identified 38,141 single nucleotide polymorphisms (SNPs) in these unigenes. Of these SNPs, 26,823 (70.3%) were found in a predicted open reading frame (ORF) and 6,998 (18.3%) were nonsynonymous. Conclusions The assembled transcriptome and SNP data are essential genomic resources for further study of the developmental pathways, genetic mechanisms and metabolic consequences of adaptive divergence in dispersal power in natural populations.

Noninvasive prenatal testing using a novel analysis pipeline to screen for all autosomal fetal aneuploidies improves pregnancy management.

Noninvasive prenatal testing by massive parallel sequencing of maternal plasma DNA has rapidly been adopted as a mainstream method for detection of fetal trisomy 21, 18 and 13. Despite the relative high accuracy of current NIPT testing, a substantial number of false-positive and false-negative test results remain. Here, we present an analysis pipeline, which addresses some of the technical as well as the biologically derived causes of error. Most importantly, it differentiates high z-scores due to fetal trisomies from those due to local maternal CNVs causing false positives. This pipeline was retrospectively validated for trisomy 18 and 21 detection on 296 samples demonstrating a sensitivity and specificity of 100%, and applied prospectively to 1350 pregnant women in the clinical diagnostic setting with a result reported in 99.9% of cases. In addition, values indicative for trisomy were observed two times for chromosome 7 and once each for chromosomes 15 and 16, and once for a segmental trisomy 18. Two of the trisomies were confirmed to be mosaic, one of which contained a uniparental disomy cell line. As placental trisomies pose a risk for low-grade fetal mosaicism as well as uniparental disomy, genome-wide noninvasive aneuploidy detection is improving prenatal management.

Differential modes of selection on the rhodopsin gene in coastal Baltic and North Sea populations of the sand goby, Pomatoschistus minutus

An excellent model to elucidate the mechanisms and importance of evolution in the marine environment is the spectral tuning mechanism of the visual pigment in vertebrates. In the sand goby Pomatoschistus minutus (Teleostei; Gobiidae), a distribution‐wide study showed that spatial variation at the rhodopsin gene (RH1) matches the characteristics of specific light environments. This match suggests that populations are locally adapted to selective light regimes targeting the RH1 gene. If so, then the direction of selection should depend on the regional spatial and temporal stability of the light conditions. We tested this prediction by comparing goby populations from two regions: the Baltic Sea, characterized by divergent, but temporally stable light conditions, and the North Sea, characterized by locally heterogeneous and temporally variable light conditions. RH1 sequences of 491 Pomatoschistus minutus individuals from 15 locations were analysed. We found that variation at the RH1 gene in the Baltic populations showed signatures of diversifying selection, whereas the RH1 gene in the North Sea showed signatures of stabilizing selection. These different modes of selection are consistent with the regional light conditions and hence support our predictions, but may also be influenced by migration between the open sea and more turbid estuarine environments. An interesting observation is that within one gene, synonymous and non‐synonymous SNPs show a totally different pattern between populations. Population differentiation based on non‐synonymous SNPs of the RH1 gene correlated with spectral variation of the local environment of the sand goby populations. In contrast, the differentiation based on synonymous SNPs of RH1 reflects more the neutral historical pattern of the species.

Annotate-it: a Swiss-knife approach to annotation, analysis and interpretation of single nucleotide variation in human disease

The increasing size and complexity of exome/genome sequencing data requires new tools for clinical geneticists to discover disease-causing variants. Bottlenecks in identifying the causative variation include poor cross-sample querying, constantly changing functional annotation and not considering existing knowledge concerning the phenotype. We describe a methodology that facilitates exploration of patient sequencing data towards identification of causal variants under different genetic hypotheses. Annotate-it facilitates handling, analysis and interpretation of high-throughput single nucleotide variant data. We demonstrate our strategy using three case studies. Annotate-it is freely available and test data are accessible to all users at http://www.annotate-it.org.

Molecular Analysis of the Breast Cancer Genes BRCA1 and BRCA2 Using Amplicon-Based Massive Parallel Pyrosequencing

The aim of this study was to implement the massively parallel sequencing technology for diagnostic applications. We evaluated an amplicon-based method for the analysis of the BRCA1 and BRCA2 genes on the Roche 454 GS-FLX sequencer, to identify disease-causing mutations in breast and/or ovarian cancer patients. A first evaluation relied on the analysis of DNA fragments containing known mutations. Secondly, the entire coding regions of the BRCA1 and BRCA2 genes were interrogated in more than 400 patient samples, using a multiplex PCR-based assay. Variants were filtered on the basis of their frequency (20%) and sequencing depth (>25×). Special attention was given to sequencing accuracy in homopolymers. In the initial evaluation, all known heterozygous mutations were detected. The percentage of mutant reads ranged from 22% to 62%. For the multiplex assay, 95% sensitivity and 91% specificity were obtained. In addition, we were able to reliably distinguish mutations from noise through the analysis of the raw signal intensities in homopolymers. This work presents an evaluation of the next-generation sequencing for use in diagnostics, based on a relatively high number of samples and experiments. We anticipate that the technique would further improve, and would allow reducing the costs per analysis and the turn-around time, to benefit patients who undergo BRCA molecular testing.

Multilocus analyses of an Antarctic fish species flock (Teleostei, Notothenioidei, Trematominae): Phylogenetic approach and test of the early-radiation event

Clades that have undergone episodes of rapid cladogenesis are challenging from a phylogenetic point of view. They are generally characterised by short or missing internal branches in phylogenetic trees and by conflicting topologies among individual gene trees. This may be the case of the subfamily Trematominae, a group of marine teleosts of coastal Antarctic waters, which is considered to have passed through a period of rapid diversification. Despite much phylogenetic attention, the relationships among Trematominae species remain unclear. In contrast to previous studies that were mostly based on concatenated datasets of mitochondrial and/or single nuclear loci, we applied various single-locus and multilocus phylogenetic approaches to sequences from 11 loci (eight nuclear) and we also used several methods to assess the hypothesis of a radiation event in Trematominae evolution. Diversification rate analyses support the hypothesis of a period of rapid diversification during Trematominae history and only a few nodes in the hypothetical species tree were consistently resolved with various phylogenetic methods. We detected significant discrepancies among trees from individual genes of these species, most probably resulting from incomplete lineage sorting, suggesting that concatenation of loci is not the most appropriate way to investigate Trematominae species interrelationships. These data also provide information about the possible effects of historic climate changes on the diversification rate of this group of fish.

Genetic diversity and population history of the migratory catfishes Pangasianodon hypophthalmus and Pangasius bocourti in the Cambodian Mekong River

Polymerase chain reaction (PCR) restriction fragment length polymorphism analysis of mitochondrial DNA was applied to the genetic structure and evolutionary history of the more ancestral Pangasianodon hypophthalmus (n=82), and the recently speciated catfish Pangasius bocourti population structure that may result from high levels of contemporary gene flow. Genetic diversity was lower in P. hypophthalmus than in P. bocourti. However, a different evolutionary history was inferred for both species based on genealogical and demographic analyses (mismatch analysis, Tajima’s D- and Fu’s Fs-tests). The genetic profile of the more ancestral P. hypophthalmus shows indications of a recent population bottleneck, whereas the recently speciated P. bocourti shows signatures of historical population expansion. This study stresses the importance of preserving the migration routes in the Cambodian Mekong basin in order to maintain the genetic diversity and long-term integrity of both species.