Genetic Variations and a Haplotype Associated with Neonatal Hyperbilirubinemia in Indonesian Population.

Neonatal hyperbilirubinemia (NH) is a common finding in newborn babies in Indonesia. Common and rare variants of have been known to contribute to NH etiology. This study aims to identify genetic variation and haplotype associated with NH in Indonesian population.

Investigation of the spatial structure and interactions of the genome at sub-kilobase-pair resolution using T2C.

Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes.

Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI.

DNA methylation is a well-known epigenetic modification that plays a crucial role in gene regulation, but genome-wide analysis of DNA methylation remains technically challenging and costly. DNA methylation-dependent restriction enzymes can be used to restrict CpG methylation analysis to methylated regions of the genome only, which significantly reduces the required sequencing depth and simplifies subsequent bioinformatics analysis. Unfortunately, this approach has been hampered by complete digestion of DNA in CpG methylation-dense regions, resulting in fragments that are too small for accurate mapping.

Comparison of Genome Sequences from Strains Isolated from Symptomatic and Asymptomatic Patients.

is a common cause of respiratory tract infections (RTIs) in children. We recently demonstrated that this bacterium can be carried asymptomatically in the respiratory tract of children. To identify potential genetic differences between strains that are carried asymptomatically and those that cause symptomatic infections, we performed whole-genome sequence analysis of 20 strains.

Control of developmentally primed erythroid genes by combinatorial co-repressor actions.

How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state.

Proteins that bind regulatory regions identified by histone modification chromatin immunoprecipitations and mass spectrometry.

The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS).

Targeted Next Generation Sequencing reveals previously unidentified TSC1 and TSC2 mutations.

Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene.

Deciphering the RNA landscape by RNAome sequencing.

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols.

Next-generation sequencing-based genome diagnostics across clinical genetics centers: implementation choices and their effects.

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants.

Identification of microRNAs in human plasma.

In recent years, microRNAs (miRNA) have been demonstrated to be present in body fluids and may therefore serve as diagnostic markers for diseases. By characterizing miRNA profiles in plasma, a miRNA signature may potentially be developed as a diagnostic and risk assessment tool for particular (patho)physiological states. This chapter describes the isolation, purification, identification, and sequencing of human plasma miRNAs.

Targeted Chromatin Capture (T2C): a novel high resolution high throughput method to detect genomic interactions and regulatory elements.

Background: Significant efforts have recently been put into the investigation of the spatial organization and the chromatin-interaction networks of genomes. Chromosome conformation capture (3C) technology and its derivatives are important tools used in this effort. However, many of these have limitations, such as being limited to one viewpoint, expensive with moderate to low resolution, and/or requiring a large sequencing effort.

Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma.

Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss of chromosome 3.

Exome sequencing and functional analyses suggest that SIX6 is a gene involved in an altered proliferation-differentiation balance early in life and optic nerve degeneration at old age.

Primary open-angle glaucoma (POAG) is a hereditary neurodegenerative disease, characterized by optic nerve changes including increased excavation, notching and optic disc hemorrhages. The excavation can be described by the vertical cup-disc ratio (VCDR). Previously, genome-wide significant evidence for the association of rs10483727 in SIX1-SIX6 locus with VCDR and subsequent POAG was found.

Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis.

Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo.

Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to β-thalassaemia.

β-Thalassaemia is one of the most common autosomal recessive single-gene disorder worldwide, with a carrier frequency of 12% in Cyprus. Prenatal tests for at risk pregnancies use invasive methods and development of a non-invasive prenatal diagnostic (NIPD) method is of paramount importance to prevent unnecessary risks inherent to invasive methods. Here, we describe such a method by assessing a modified version of next generation sequencing (NGS) using the Illumina platform, called 'targeted sequencing', based on the detection of paternally inherited fetal alleles in maternal plasma.

Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions.

Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g.

Genome-wide analysis shows that Ldb1 controls essential hematopoietic genes/pathways in mouse early development and reveals novel players in hematopoiesis.

The first site exhibiting hematopoietic activity in mammalian development is the yolk-sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in blast colony-forming cells to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk-sac hematopoiesis and vascular development in Ldb1(-/-) mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells.

Snail regulates MyoD binding-site occupancy to direct enhancer switching and differentiation-specific transcription in myogenesis.

In skeletal myogenesis, the transcription factor MyoD activates distinct transcriptional programs in progenitors compared to terminally differentiated cells. Using ChIP-Seq and gene expression analyses, we show that in primary myoblasts, Snail-HDAC1/2 repressive complex binds and excludes MyoD from its targets. Notably, Snail binds E box motifs that are G/C rich in their central dinucleotides, and such sites are almost exclusively associated with genes expressed during differentiation.

Genome-wide DNA methylation profiling of non-small cell lung carcinomas.

Background: Non-small cell lung carcinoma (NSCLC) is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors.

Transcriptional dominance of Pax7 in adult myogenesis is due to high-affinity recognition of homeodomain motifs.

Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome-wide binding-site analysis, we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function.

Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development.

The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron.

Absence of common somatic alterations in genes on 1p and 19q in oligodendrogliomas.

A common and histologically well defined subtype of glioma are the oligodendroglial brain tumors. Approximately 70% of all oligodendrogliomas have a combined loss of the entire 1p and 19q chromosomal arms. This remarkably high frequency suggests that the remaining arms harbor yet to be identified tumor suppressor genes.

Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes.

The HMG-box transcription factor Sox2 plays a role throughout neurogenesis and also acts at other stages of development, as illustrated by the multiple organs affected in the anophthalmia syndrome caused by SOX2 mutations. Here we combined proteomic and genomic approaches to characterize gene regulation by Sox2 in neural stem cells. Chd7, a chromatin remodeling ATPase associated with CHARGE syndrome, was identified as a Sox2 transcriptional cofactor.