The zebrafish homeobox gene Hox(zf-114): primary structure, expression pattern and evolutionary aspects

It is gradually becoming accepted that vertebrate homeobox genes, like their counterparts in Drosophila, are crucial for normal development of the embryo. Most vertebrate homeoboxes reported so far are related to the Drosophila Antennapedia (Antp) sequence, and here we describe hox[zf-114], a novel Antp-like homeobox gene from the zebrafish. The sequence of the hox[zf-114] homeodomain indicates that this gene could be a member of a subfamily defined by the mouse Hox-1.5/-2.7/-4.1 genes. However, the evolutionary origin of hox[zf-114] is unclear and, based on the putative protein sequence, we conclude that it is not directly homologous to Hox-1.5, Hox-2.7 or Hox-4.1, or to other known mammalian homeobox genes. Nevertheless, as revealed by in situ hybridization, hox[zf-114] exhibits a spatial expression pattern typical for vertebrate Antp-like homeobox genes. Transcripts are detected in the posterior hindbrain, where a sharp anterior border of expression is observed, and throughout the spinal cord. The hox[zf-114] gene is also active in a region that gives rise to the pectoral fins. These findings suggest a role for hox[zf-114] in anteroposterior patterning of the neural tube and in pectoral fin development.publishedVersio

pRb2/p130 protein expression and RBL2 mutation analysis in Burkitt lymphoma from Uganda

<p>Abstract</p> <p>Background</p> <p>The members of the retinoblastoma protein family, pRb, p107 and pRb2 (p130), are central players in controlling the cell cycle. Whereas disturbed function of pRb is commonly seen in human cancers, it is still an open question whether pRb2 is involved in tumorigenic processes. However, altered subcellular localization of pRb2 and mutations in the pRb2-encoding gene <it>RBL2 </it>have been described for some tumours, including Burkitt lymphomas (BL).</p> <p>Methods</p> <p>We retrieved 51 biopsy specimens of endemic BL cases from Uganda. The expression of pRb2 was determined by immunohistochemistry. Exons 1922 of the <it>RBL2 </it>gene, the region known to contain a nuclear localization signal, were screened for mutations by PCR amplification and direct DNA sequencing.</p> <p>Results</p> <p>Nearly all of our cases (84.0%) were positive for pRb2 protein expression although this protein is a marker for growth arrest and Burkitt lymphoma is characterized by a high proliferation rate. Of the positive cases, 73.8% were scored as expressing the protein at a high level. Subcellular pRb2 localization was predominantly nuclear and no cases with expression restricted to the cytoplasm were observed. We did not detect any <it>RBL2 </it>mutations in the part of the gene that encodes the C-terminal end of the protein.</p> <p>Conclusion</p> <p>The majority of endemic BL cases from Uganda express pRb2, but somatic <it>RBL2 </it>mutations affecting the protein's nuclear localization signal appear to be rare.</p

Type 2 diabetes genes : present status and data from Norwegian studies

The worldwide rise in prevalence of type 2 diabetes has led to an intense search for the genetic risk factors of this disease. In type 2 diabetes and other complex disorders, multiple genetic and environmental factors, as well as the interaction between these factors, determine the phenotype. In this review, we summarize present knowledge, generated by more than two decades of efforts to dissect the genetic architecture of type 2 diabetes. Initial studies were either based on a candidate gene approach or attempted to fine-map signals generated from linkage analysis. Despite the detection of multiple genomic regions proposed to be linked to type 2 diabetes, subsequent positional fine-mapping of candidates were mostly inconclusive. However, the introduction of genome-wide association studies (GWAS), applied on thousands of patients and controls, completely changed the field. To date, more than 50 susceptibility loci for type 2 diabetes have been detected through the establishment of large research consortia, the application of GWAS on intermediary diabetes phenotypes and the use of study samples of different ethnicities. Still, the common variants identified in the GWAS era only explain some of the heritability seen for type 2 diabetes. Thus, focus is now shifting towards searching also for rare variants using new high-throughput sequencing technologies. For genes involved in the genetic predisposition to type 2 diabetes the emerging picture is that there are hundreds of different gene variants working in a complex interplay influencing pancreatic beta cell function/mass and, only to a lesser extent, insulin action. Several Norwegian studies have contributed to the field, extending our understanding of genetic risk factors in type 2 diabetes and in diabetes-related phenotypes like obesity and cardiovascular disease.publishedVersio

KRAS mutation analysis by droplet digital PCR of duodenal juice from patients with MODY8 and other pancreatic diseases

Background Maturity-onset diabetes of the young type 8 (MODY8 or CEL-MODY) is an inherited pancreatic disease characterized by chronic inflammation of the pancreas and diabetes. It is not known whether MODY8 patients have increased risk for developing pancreatic cancer. We investigated KRAS mutation load in duodenal juice from MODY8 patients, comparing with other groups of pancreatic disease. Methods Droplet digital PCR (ddPCR) was used to detect KRAS codon 12/13/61 mutations in duodenal juice sampled from 11 MODY8 patients, nine healthy subjects and 100 patients clinically investigated due to suspected pancreatic disease. Results KRAS mutations were detected in 4/11 patients with MODY8 (36%), 1/9 healthy subjects (11%), 15/44 patients with chronic pancreatitis (CP, 34%), 3/5 patients with pancreatic ductal adenocarcinoma (PDAC, 60%), 3/20 patients with acute pancreatitis (15%), 0/13 patients with other pancreatic disorders and 2/18 patients with nonpancreatic gastrointestinal disease (11%). Of the 28 positive juice samples, 25 (89%) had low-abundance mutations in codons 12/13, with a variant allele frequency (VAF) less than 1%. KRAS-positive patients with MODY8 or CP had significantly lower VAFs than patients with PDAC (Mann-Whitney U test; p = 0.041). Although the overall mutation detection rate was higher for subjects ≥50 years old (26%) than for younger subjects (15%), the difference was not statistically significant. Conclusions KRAS mutations were detectable in duodenal juice from MODY8 patients, but with low abundance and at the same frequency as in CP patients. The discriminative value of the analysis with regard to other pancreatic disease was limited.publishedVersio

Novel hybridization- and tag-based error-corrected method for sensitive ctDNA mutation detection using ion semiconductor sequencing

Circulating tumor DNA (ctDNA) analysis has emerged as a clinically useful tool for cancer diagnostics and treatment monitoring. However, ctDNA detection is complicated by low DNA concentrations and technical challenges. Here we describe our newly developed sensitive method for ctDNA detection on the Ion Torrent sequencing platform, which we call HYbridization- and Tag-based Error-Corrected sequencing (HYTEC-seq). This method combines hybridization-based capture with molecular tags, and the novel variant caller PlasmaMutationDetector2 to eliminate background errors. We describe the validation of HYTEC-seq using control samples with known mutations, demonstrating an analytical sensitivity down to 0.1% at > 99.99% specificity. Furthermore, to demonstrate the utility of this method in a clinical setting, we analyzed plasma samples from 44 patients with advanced pancreatic cancer, revealing mutations in 57% of the patients at allele frequencies as low as 0.23%.publishedVersio

Tissue MicroRNA profiles as diagnostic and prognostic biomarkers in patients with resectable pancreatic ductal adenocarcinoma and periampullary cancers

BACKGROUND: The aim of this study was to validate previously described diagnostic and prognostic microRNA expression profiles in tissue samples from patients with pancreatic cancer and other periampullary cancers. METHODS: Expression of 46 selected microRNAs was studied in formalin-fixed paraffin-embedded tissue from patients with resected pancreatic ductal adenocarcinoma (n = 165), ampullary cancer (n=59), duodenal cancer (n = 6), distal common bile duct cancer (n = 21), and gastric cancer (n = 20); chronic pancreatitis (n = 39); and normal pancreas (n = 35). The microRNAs were analyzed by PCR using the Fluidigm platform. RESULTS: Twenty-two microRNAs were significantly differently expressed in patients with pancreatic cancer when compared to healthy controls and chronic pancreatitis patients; 17 miRNAs were upregulated (miR-21-5p, −23a-3p, −31-5p, −34c-5p, −93-3p, −135b-3p, −155-5p, −186-5p, −196b-5p, −203, −205-5p, −210, −222-3p, −451, −492, −614, and miR-622) and 5 were downregulated (miR-122-5p, −130b-3p, −216b, −217, and miR-375). MicroRNAs were grouped into diagnostic indices of varying complexity. Ten microRNAs associated with prognosis were identified (let-7 g, miR-29a-5p, −34a-5p, −125a-3p, −146a-5p, −187, −205-5p, −212-3p, −222-5p, and miR-450b-5p). Prognostic indices based on differences in expression of 2 different microRNAs were constructed for pancreatic and ampullary cancer combined and separately (30, 5, and 21 indices). CONCLUSION: The study confirms that pancreatic cancer tissue has a microRNA expression profile that is different from that of other periampullary cancers, chronic pancreatitis, and normal pancreas. We identified prognostic microRNAs and microRNA indices that were associated with shorter overall survival in patients with radically resected pancreatic cancer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40364-017-0087-6) contains supplementary material, which is available to authorized users

GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation

AbstractGCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically ‘normal’ GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations

Pathogenic Carboxyl Ester Lipase (CEL) Variants Interact with the Normal CEL Protein in Pancreatic Cells

Mutations in the gene encoding the digestive enzyme carboxyl ester lipase (CEL) are linked to pancreatic disease. The CEL variant denoted CEL-HYB predisposes to chronic pancreatitis, whereas the CEL-MODY variant causes MODY8, an inherited disorder of endocrine and exocrine pancreatic dysfunction. Both pathogenic variants exhibit altered biochemical and cellular properties compared with the normal CEL protein (CEL-WT, wild type). We here aimed to investigate effects of CEL variants on pancreatic acinar and ductal cell lines. Following extracellular exposure, CEL-HYB, CEL-MODY, and CEL-WT were endocytosed. The two pathogenic CEL proteins significantly reduced cell viability compared with CEL-WT. We also found evidence of CEL uptake in primary human pancreatic acinar cells and in native ductal tissue. Moreover, coexpression of CEL-HYB or CEL-MODY with CEL-WT affected secretion of the latter, as CEL-WT was observed to accumulate intracellularly to a higher degree in the presence of either pathogenic variant. Notably, in coendocytosis experiments, both pathogenic variants displayed a modest effect on cell viability when CEL-WT was present, indicating that the normal protein might diminish toxic effects conferred by CEL-HYB and CEL-MODY. Taken together, our findings provide valuable insight into how the pathogenic CEL variants predispose to pancreatic disease and why these disorders develop slowly over time.publishedVersio

Integrin α11β1 is expressed in breast cancer stroma and associates with aggressive tumor phenotypes

Cancer‐associated fibroblasts are essential modifiers of the tumor microenvironment. The collagen‐binding integrin α11β1 has been proposed to be upregulated in a pro‐tumorigenic subtype of cancer‐associated fibroblasts. Here, we analyzed the expression and clinical relevance of integrin α11β1 in a large breast cancer series using a novel antibody against the human integrin α11 chain. Several novel monoclonal antibodies against the integrin α11 subunit were tested for use on formalin‐fixed paraffin‐embedded tissues, and Ab 210F4B6A4 was eventually selected to investigate the immunohistochemical expression in 392 breast cancers using whole sections. mRNA data from METABRIC and co‐expression patterns of integrin α11 in relation to αSMA and cytokeratin‐14 were also investigated. Integrin α11 was expressed to varying degrees in spindle‐shaped cells in the stroma of 99% of invasive breast carcinomas. Integrin α11 co‐localized with αSMA in stromal cells, and with αSMA and cytokeratin‐14 in breast myoepithelium. High stromal integrin α11 expression (66% of cases) was associated with aggressive breast cancer features such as high histologic grade, increased tumor cell proliferation, ER negativity, HER2 positivity, and triple‐negative phenotype, but was not associated with breast cancer specific survival at protein or mRNA levels. In conclusion, high stromal integrin α11 expression was associated with aggressive breast cancer phenotypes.publishedVersio

Deficiency of the metabolic enzyme SCHAD in pancreatic β-cells promotes amino acid–sensitive hypoglycemia

Congenital hyperinsulinism of infancy (CHI) can be caused by a deficiency of the ubiquitously expressed enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD). To test the hypothesis that SCHAD-CHI arises from a specific defect in pancreatic β-cells, we created genetically engineered β-cell-specific (β-SKO) or hepatocyte-specific (L-SKO) SCHAD knockout mice. While L-SKO mice were normoglycemic, plasma glucose in β-SKO animals was significantly reduced in the random-fed state, after overnight fasting, and following refeeding. The hypoglycemic phenotype was exacerbated when the mice were fed a diet enriched in leucine, glutamine, and alanine. Intraperitoneal injection of these three amino acids led to a rapid elevation in insulin levels in β-SKO mice compared to controls. Consistently, treating isolated β-SKO islets with the amino acid mixture potently enhanced insulin secretion compared to controls in a low-glucose environment. RNA sequencing of β-SKO islets revealed reduced transcription of β-cell identity genes and upregulation of genes involved in oxidative phosphorylation, protein metabolism, and Ca2+ handling. The β-SKO mouse offers a useful model to interrogate the intra-islet heterogeneity of amino acid sensing given the very variable expression levels of SCHAD within different hormonal cells, with high levels in β- and δ-cells and virtually absent α-cell expression. We conclude that the lack of SCHAD protein in β-cells results in a hypoglycemic phenotype characterized by increased sensitivity to amino acid-stimulated insulin secretion and loss of β-cell identity.publishedVersio