Whole-genome sequencing analysis of the cardiometabolic proteome.

The human proteome is a crucial intermediate between complex diseases and their genetic and environmental components, and an important source of drug development targets and biomarkers. Here, we comprehensively assess the genetic architecture of 257 circulating protein biomarkers of cardiometabolic relevance through high-depth (22.5×) whole-genome sequencing (WGS) in 1328 individuals. We discover 131 independent sequence variant associations (P < 7.45 × 10-11) across the allele frequency spectrum, all of which replicate in an independent cohort (n = 1605, 18.4x WGS). We identify for the first time replicating evidence for rare-variant cis-acting protein quantitative trait loci for five genes, involving both coding and noncoding variation. We construct and validate polygenic scores that explain up to 45% of protein level variation. We find causal links between protein levels and disease risk, identifying high-value biomarkers and drug development targets

Biomarkers and viral risk factors in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease resulting in axonal injury and demyelination of nerve fibers in the central nervous system. The causal factor and pathological mechanisms governing disease progression have remained elusive, limiting the available clinical tools for MS diagnosis and treatment monitoring, particularly in the periphery. In the following studies, we investigated potential [1] protein biomarkers capable of differentiating MS and characterizing disease activity and [2] serological biomarkers that may predict MS risk or adverse events associated with immunomodulatory treatment. Studies I-III measured proteins in cerebrospinal fluid (CSF) and plasma using a highsensitivity proximity extension immunoassay to characterize the differential protein profile associated with MS. Although many require further validation, several proteins in CSF (e.g., IL12B and CD79B) and plasma (e.g., OSM) show promise in differentiating early stages of MS and assessing progressive disease, relapse activity, and severity of disability progression. Many also complement current diagnostic tools for MS, including oligoclonal bands, IgG index, and MRI T2 lesions. Measures were responsive to common disease-modifying drugs, including natalizumab and fingolimod, indicating the potential for monitoring treatment efficacy. Lastly, in Study IV, we characterized the susceptibility of several of these inflammation-related proteins to sample processing conditions, a necessary consideration for reliable usage in clinical practice. In Study V, we showed high anti-EBNA1 IgG levels (Epstein-Barr virus, EBV), particularly against the aa385-420 peptide fragment, were associated with an increased risk for MS, independent of any history of infectious mononucleosis. Genome-wide association analysis showed HLA as the primary genetic risk factor for high EBNA-1/VCAp18 antibody response, particularly the MS risk haplotype with DRB1*15:01. A bi-directional relationship between EBV and MS genetic susceptibility was observed, with genetic susceptibility to high EBNA- 1 IgG resulting in an increased risk for MS and vice versa. Overlapping HLA associations with MS and previous auto-antigen studies may indicate the role of molecular mimicry. Study VI identified genetic risk factors affecting JC polyomavirus (JCV) seropositivity, a risk assessment measure for progressive multifocal leukoencephalopathy (PML). In addition to HLA, genetic polymorphisms in the FUT2 and ABO genes, corresponding with secretor status and blood group phenotypes, were associated with anti-JCV(VP1) IgG levels. These findings indicate a potential neutralization effect of ABH antigens against JCV. However, further investigation is needed to assess the implications of HLA and blood phenotypes in the risk assessment of PML, particularly for MS treatment

Colorectal adenoma and carcinoma specific miRNA profiles in biopsy and their expression in plasma specimens

BACKGROUND: MiRNA expression markers are well characterized in colorectal cancer (CRC), but less is known about miRNA expression profiles in colorectal adenomas. Genome-wide miRNA and mRNA expression analyses were conducted through the colorectal adenoma dysplasia sequence. Furthermore, analysis of the expression levels of miRNAs in matched plasma samples was performed, focusing on biomarker candidates; miRNA and mRNA expression analyses were performed on colorectal biopsies and plasma samples (20 normals; 11 tubular and 9 tubulovillous adenomas; 20 colorectal carcinomas) by miRNA 3.0 and Human Transcriptome Array (Affymetrix) and validated by RT-qPCR. Microarray data were analyzed using Expression Console and mRNA targets were predicted using miRWALK 2.0. RESULTS: Based on microarray analysis, 447 miRNAs were expressed in tissue and 320 in plasma. Twelve were upregulated (miR-31, 8-fold p < 0.001) and 11 were downregulated (miR-10b 3-fold p < 0.001) in neoplastic lesions compared to normal group. Eleven miRNAs showed altered expression between adenoma subtypes (miR-183 2.8-fold change, p < 0.007). Expression level of 24 miRNAs differed between adenoma and CRC groups (including miR-196a, 3.5-fold). Three miRNAs (miR-31, miR-4506, miR-452*) were differentially expressed in adenoma compared to normal both in tissue and plasma samples. miRNA expression data were confirmed by RT-PCR both in plasma and matched tissue samples. CONCLUSIONS: MiRNAs showed characteristic expression changes during CRC development in tissue. miRNAs were also presented in plasma and positively correlated with matched tissue expression levels. The identified miRNA expression changes could be verified RT-PCR methods facilitating routine application. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13148-016-0305-3) contains supplementary material, which is available to authorized users

The multifaceted role of mutant p53 in remodeling the tumor transcriptome and secretome

The tumor suppressor gene TP53 is frequently inactivated by missense mutations in human cancers. These genetic alterations give rise to the expression of mutant variants of the encoded p53 protein (mutp53) that have lost their tumor-suppressive functions. Moreover, many mutp53 proteins acquire novel neomorphic properties that can significantly modulate cancer cell behavior, promoting aggressiveness, metastasis, and therapy resistance. Mutp53 primarily promotes tumor growth by interacting with various cellular proteins, especially transcription factors, leading to significant changes in transcriptional regulation. Remarkably, these pro-tumorigenic molecular alterations are not limited to intracellular processes but may also extend to the extracellular environment through mutp53-mediated changes in the composition of the tumor cell secretome. Consequently, this can profoundly influence the tumor microenvironment, creating a supportive milieu that favors tumor progression. The exceptionally high prevalence of TP53 mutations in human tumors and the diverse pro-tumorigenic activities of mutp53 render it an attractive target for cancer therapy. However, despite extensive research, no drug designed to specifically target tumors with TP53 mutations has been approved for clinical use, underscoring the ongoing need for further research into the underlying mechanisms. In this study, various cancer models were established and characterized to enhance our understanding of mutp53's role in shaping the tumor microenvironment. Different human and murine cancer cell lines that harbor TP53 mutations were successfully deleted of mutp53 using the CRISPR/Cas9 technology. In addition, a conditional mutp53 knockout cell line was generated, enabling the acute deletion of mutp53. This approach facilitated the study of mutp53's oncogenic functions in a cell line highly dependent on mutp53. Furthermore, primary cell cultures were derived from paired cohorts of mice with mutp53-driven and mutp53-deficient small cell and non-small cell lung cancer tumors, induced by a combination of germline and somatic, CRISPR/Cas9-mediated, in vivo gene editing. Phenotypic analyses of small cell lung cancer derived cell cultures unveiled that mutp53 plays a role in enforcing the neuroendocrine phenotype, holding significant therapeutic implications. Moreover, transcriptomic profiling of all the generated mutp53-deleted tumor cell cultures, along with their parental mutp53-expressing counterparts, revealed several secreted factors and signaling pathways differentially regulated by mutp53. Notably, mutp53 consistently suppressed proinflammatory signaling pathways across various cancer models. Intriguingly, targeted proteomic analysis of the tumor cell secretome indicated that mutp53 stimulates global protein secretion and induces the context-specific secretion of several key components of the NF-κB signaling pathway, with NF-κB coactivator MTDH being most prominent. In summary, the generation and combined transcriptomic and proteomic analysis of isogenic cell-based cancer models have provided insights into mutp53-regulated factors and signaling pathways that participate in the reciprocal communication between cancer cells and their surroundings. This work highlights the multifaceted potential of mutp53 in remodeling the tumor microenvironment

Understanding the role of osteoclasts in wild-type craniofacial development and genetic mapping of skeletal mutants in zebrafish

Detailed understanding of skeletal development, especially in the craniofacial space, requires close observation of growth patterns along with understanding of the genetics involved. Prior work on craniofacial development has focused on imaging data taken at specific timepoints on fixed specimens. The zebrafish offers advantages as a model organism to address the extant challenges in this field of research as many aspects of skull formation and general skeletal development are conserved from zebrafish to mammals. Zebrafish remain optically clear well into the juvenile stage, encompassing the core period of bone development in the skull. Taking advantage of many fluorescent transgenic lines marking cells that contribute to skeletal development, our lab has developed protocols to perform confocal live imaging multiple times on individual fish. I have used those imaging protocols for two main objectives: 1) to establish atlases of tissue patterning during cranial development; and 2) to characterize craniofacial and general skeletal development in mutants identified through forward genetic screens. Using serial low-magnification confocal imaging, fish carrying transgenic markers for osteoblasts (sp7:mCherry) and chondrocytes (col1a1:eGFP) were tracked for approximately 30 days, focusing on bones of the skull vault. We defined three stages of growth: initiation by osteoblasts on a cartilage scaffold; a period of rapid planar growth by the frontal and parietal bones; and a plateauing of growth and maturation of the sutures. We captured confocal Z-stacks which were converted into 3D models. To further understand the complex cell interactions of craniofacial development, we used a transgene marking osteoclasts, bone-resorbing cells which represent the innate immune component within the bone. Osteoclasts are critical in homeostasis of adult skeletal structures, but there is limited information on the role of osteoclasts in craniofacial development. We used confocal microscopy of live transgenic fish to determine the location of osteoclasts in the developing skull daily over several weeks, from 5.18 mm to 9.6 mm standard length (approximately 15 to 34 days post fertilization). The overall distribution of osteoclasts is consistent among individuals, but they are sparse in most areas of the skull and the exact locations vary among fish and across developmental time points. We observed osteoclasts concentrating at areas of remodeling associated with neuromasts and their associated nerves, the hyomandibular foramina and around the supraorbital lateral line. This suggests that they play a special role in bone remodeling around neuromasts and nerves during craniofacial development. In csfr1amh5 mutants lacking functional osteoclasts, the morphology of the cranial bone was disrupted in both areas. The hyomandibular foramen is present in the initial cartilage template, but after ossification has begun, the diameter of the canal is significantly smaller in the absence of osteoclasts. The diameter of the supraorbital lateral line canals was also reduced in the mutants, but the more significant difference was the paucity of small foramina in and around the canals, which allow passage of efferent nerves through the bone. These findings define important and previously unappreciated roles for osteoclast activity during craniofacial development. With this data on baseline developmental benchmarks and tissue patterning in wild-type individuals we then focused on two skeletal mutants, toth and jinx, identified in a genetic screen. toth was originally identified due to severely lagging growth and structural skull abnormalities, including numerous pits in the frontal and parietal bones. Mutants for toth have a dramatic accumulation of osteoclasts on the frontal and parietal bones corresponding to the pits in the bone. jinx mutants have craniofacial irregularities but have additional skeletal abnormalities, including sporadic fusion of vertebral bodies. Using the sp7:mcherry transgenic line and fluorescent vital staining of mineralized bone, I have documented uneven mineralization and vertebral fusion events in live jinx mutants during development. We have localized the genes for toth and jinx based on filtering bulked sequencing for single nucleotide polymorphisms. I have further refined the mapping of jinx using microsatellite markers to implicate the candidate gene entpd5a, which has previously been implicated in bone mineralization in zebrafish. I am performing additional experiments to verify the gene identity and mechanisms underlying the defects. My thesis presents a body of work demonstrating the power of live imaging in zebrafish to reveal developmental processes. We created an atlas of bone and cartilage morphology during normal skull development and building on that foundation demonstrated previously unknown requirements for osteoclasts in sculpting skull morphology. We have applied the same imaging tools to characterize developmental processes in two zebrafish mutants with craniofacial and skeletal defects, and in future experiments will be able to correlate those defects with specific genetic lesions

Inflammatory bowel disease:The genetic background and beyond

Inflammatoire darm ziekten (IBD) zoals de ziekte van Crohn (CD) en colitis ulcerosa (CU) zijn deels erfelijk en deels omgevingsbepaalde ziekten en worden gekenmerkt door een chronisch recidiverende ontsteking van de darm. Waarschijnlijk zijn een verstoorde immuunreactie op normale darmflora samen met een verminderde darmwand integriteit onderliggend aan deze soms zeer ernstige en hardnekkige ziekte. Meer inzicht in de pathogenese is derhalve essentieel. Door het DNA te screenen op risico gebieden, zijn er tot op heden 163 gebieden gevonden geassocieerd met een verhoogd ziekte risico. Hiermee kan 15-25% van de totale erfelijkheid verklaard worden. In dit proefschrift worden een aantal potentiële bronnen van de ontbrekende erfelijkheid onderzocht. Er zouden meer risico gebieden kunnen zijn voor IBD. Wij analyseerden een aantal strategisch gekozen gebieden en vonden twee nieuwe risico gebieden voor IBD, en we vonden een omgekeerde associatie voor een specifiek gen voor CD en CU. Verder kan er mogelijk meer van de erfelijkheid verklaard worden met bekende risico gebieden. Mogelijk heeft dezelfde genetisch code een ander effect op ziekte risico als deze van vader of moeder wordt geërfd. Wij testten de overlappende IBD risico gebieden en vonden hier weinig aanwijzingen voor. Daarnaast werd onderzocht wat de effecten van de hoeveelheid risico gebieden is op cel niveau in afweer cellen. Deze relatie hebben wij niet kunnen vinden, wel vonden we een verschil tussen gezonde mensen en patiënten. Wij hebben bijgedragen aan de identificatie van nieuwe risico gebieden op het DNA voor inflammatoire darmziekten en potentiële bronnen voor de ontbrekende erfelijkheid onderzocht.Inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) are partially heritable and partially environmentally determined diseases and are characterized by a chronically relapsing inflammation of the gut. Probably an aberrant immune response to commensal microflora of the bowel together with a diminished integrity of the wall is culprit to this serious disease. More insight in the pathogenesis is essential. By screening DNA for risk areas, there are up until now 163 of such areas associated to increased disease risk, with which we can explain 15-25% of the heritability. In this thesis we investigate a number of potential sources of this hidden heritability. There might be more risk areas for IBD. We analyzed a number of strategically selected areas and identified two new risk areas for IBD, in another study we found altered risk for a UC and CD gene. Further, more of the heritability might be explained with the known risk areas. Potentially the same genetic code has a different effect to disease risk when inherited from the father or the mother. We tested the overlapping IBD risk areas and found limited evidence for such effects.Further we investigated what the effects of a number of risk areas is on a cellular level in immune cells. We could not find this relation, however we found a difference between healthy individuals and patients.We contributed to the identification of new risk areas on the DNA for IBD and investigated other potential sources for the hidden heritability

Characterizing Thyroid Hormone Mediated Action on Gene Expression in Mice: Mechanistic Insight into Thyroid Hormone Response Elements, Thyroid Hormone Receptor-binding Sites, and MicroRNAs

Thyroid hormone (TH) exerts its effects by binding to the TH receptor (TR), which binds to TH response elements (TREs) to regulate target gene expression. Disruption of TH action can have detrimental health effects. The precise molecular mechanisms involved in TH mediated gene expression remain unclear. The overall objectives of this thesis were to: i) characterize global gene and microRNA (miRNA) expression in early response to TH perturbation in mouse liver; ii) identify TREs and TR-binding sites found throughout the mouse genome; and iii) compare TRE half-site organizations and their ability to drive gene expression. Transcriptional profiling of mRNA liver samples from TH disrupted mice enabled the identification of genes that were under direct TH-regulation. TREs in the promoter region of Tor1a, Hectd3, Slc25a45 and 2310003H01Rik were validated in vitro, adding four genes to the battery of only 13 known TRE-containing mouse genes. Hepatic miRNAs were also found to be significantly altered following perturbations in TH levels. In vitro analyses confirmed TH regulation of miR-206. Moreover, Mup1 and Gpd2 were confirmed to be targeted by miR-206 in response to TH, demonstrating that miRNAs can act as master regulators of the TH response pathway. ChIP-chip analysis identified TR-DNA interactions in juvenile mouse liver revealing only a few TR-binding sites consistent between all analyzed samples, suggesting that relatively few genes are under direct TH/TR control. Reporter assays confirmed the presence of TREs in the promoter regions of Ddx54 and Thrsp, thus validating two additional functional mouse TREs. Finally, we investigated the relative ability of liganded homodimers of TR and retinoid X receptor (RXR), and the heterodimer TR/RXR, to regulate gene expression for three TRE half-site organizations. We found that there were fundamental differences between TRE configurations that affect nuclear receptor interactions with the response element and the ability to specifically bind their ligands. These studies provide mechanistic insight into TREs, TR-binding sites and TH action. Collectively this thesis increases our understanding of how TH operates to control genome function and provides a basis to develop appropriate testing strategies for environmental chemicals that may disrupt TH-associated genes expression by interaction with TR