Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury. Conclusion In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

The mutation causing the black-and-tan pigmentation phenotype of Mangalitza pigs maps to the porcine ASIP locus but does not affect its coding sequence

The gene for agouti signaling protein (ASIP) is centrally involved in the expression of coat color traits in animals. The Mangalitza pig breed is characterized by a black-and-tan phenotype with black dorsal pigmentation and yellow or white ventral pigmentation. We investigated a Mangalitza x Piétrain cross and observed a coat color segregation pattern in the F2 generation that can be explained by virtue of two alleles at the MC1R locus and two alleles at the ASIP locus. Complete linkage of the black-and-tan phenotype to microsatellite alleles at the ASIP locus on SSC 17q21 was observed. Corroborated by the knowledge of similar mouse coat color mutants, it seems therefore conceivable that the black-and-tan pigmentation of Mangalitza pigs is caused by an ASIP allele a(t), which is recessive to the wild-type allele A. Toward positional cloning of the a(t) mutation, a 200-kb genomic BAC/PAC contig of this chromosomal region has been constructed and subsequently sequenced. Full-length ASIP cDNAs obtained by RACE differed in their 5' untranslated regions, whereas they shared a common open reading frame. Comparative sequencing of all ASIP exons and ASIP cDNAs between Mangalitza and Piétrain pigs did not reveal any differences associated with the coat color phenotype. Relative qRT-PCR analyses showed different dorsoventral skin expression intensities of the five ASIP transcripts in black-and-tan Mangalitza. The a(t) mutation is therefore probably a regulatory ASIP mutation that alters its dorsoventral expression pattern.

Distribution of mRNA coding for 5-hydroxytryptamine receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 7 subtypes of 5-hydroxytryptamine receptors (5-HTRs) in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation (CDD). SAMPLE POPULATION: Full-thickness intestinal wall biopsy specimens were obtained from the ileum, cecum, proximal loop of the ascending colon, and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy dairy cows allocated to 2 control groups (specimens collected during routine laparotomy [group 2] or after cows were slaughtered [group 3]). PROCEDURE: Amounts of mRNA coding for 7 subtypes of 5-HTRs (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, and 5-HT4) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed as the percentage of mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA coding for 5-HTR1B, 5-HTR2B, and 5-HTR4 was significantly lower in cows with CDD than in healthy cows. For 5-HTR2B and 5-HTR4, significant differences between cows with CDD and control cows were most pronounced for the ELSC. Expression of mRNA for 5-HTR1D, 5-HTR1F, and 5-HTR2A was extremely low in all groups, and mRNA for 5-HTR1A was not detected. CONCLUSIONS AND CLINICAL RELEVANCE: Relative concentrations of mRNA coding for 5-HTR1B, 5-HT2B, and 5-HTR4 were significantly lower in the intestines of cows with CDD than in the intestines of healthy dairy cows, especially for 5-HT2B and 5-HTR4 in the ELSC. This supports the hypothesis that serotonergic mechanisms, primarily in the spiral colon, are implicated in the pathogenesis of CDD.

Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene

The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.

Comparison of musical activities of cochlear implant users with different speech-coding strategies

Speech coding might have an impact on music perception of cochlear implant users. This questionnaire study compares the musical activities and perception of postlingually deafened cochlear implant users with three different coding strategies (CIS, ACE, SPEAK) using the Munich Music Questionnaire. Overall, the self-reported perception of music of CIS, SPEAK, and ACE users did not differ by very much.

The end1 gene of Schizosaccharomyces pombe coding for a DNase is identical with the pnu1 gene coding for an RNase

The DNA nuclease activity encoded by the end1 gene, and its inactivation by mutation, was described in connection with the characterization of DNA topoisomerases in the fission yeast Schizosaccharomyces pombe (Uemura and Yanagida, 1984). Subsequently, end1 mutant strains were used for the preparation of cell extracts for the study of enzymes and intermediates involved in DNA metabolism. The molecular identification of the end1 gene and its identity with the pnu1 gene is presented. The end1-458 mutation alters glycine to glutamate in the conserved motif TGPYLP. The pnu1 gene codes for an RNase that is induced by nitrogen starvation (Nakashima et al., 2002b). Thus, the End1/Pnu1 protein, like related mitochondrial proteins in other organisms, is an example of a sugar-non-specific nuclease. The analysis of strains carrying a pnu1 deletion revealed no defects in meiotic recombination and spore viability.

Impact of unlinked deaths and coding changes on mortality trends in the Swiss National Cohort

BACKGROUND Results of epidemiological studies linking census with mortality records may be affected by unlinked deaths and changes in cause of death classification. We examined these issues in the Swiss National Cohort (SNC). METHODS The SNC is a longitudinal study of the entire Swiss population, based on the 1990 (6.8 million persons) and 2000 (7.3 million persons) censuses. Among 1,053,393 deaths recorded 1991-2007 5.4% could not be linked using stringent probabilistic linkage. We included the unlinked deaths using pragmatic linkages and compared mortality rates for selected causes with official mortality rates. We also examined the impact of the 1995 change in cause of death coding from version 8 (with some additional rules) to version 10 of the International Classification of Diseases (ICD), using Poisson regression models with restricted cubic splines. Finally, we compared results from Cox models including and excluding unlinked deaths of the association of education, marital status, and nationality with selected causes of death. RESULTS SNC mortality rates underestimated all cause mortality by 9.6% (range 2.4%-17.9%) in the 85+ population. Underestimation was less pronounced in years nearer the censuses and in the 75-84 age group. After including 99.7% of unlinked deaths, annual all cause SNC mortality rates were reflecting official rates (relative difference between -1.4% and +1.8%). In the 85+ population the rates for prostate and breast cancer dropped, by 16% and 21% respectively, between 1994 and 1995 coincident with the change in cause of death coding policy. For suicide in males almost no change was observed. Hazard ratios were only negligibly affected by including the unlinked deaths. A sudden decrease in breast (21% less, 95% confidence interval: 12%-28%) and prostate (16% less, 95% confidence interval: 7%-23%) cancer mortality rates in the 85+ population coincided with the 1995 change in cause of death coding policy. CONCLUSIONS Unlinked deaths bias analyses of absolute mortality rates downwards but have little effect on relative mortality. To describe time trends of cause-specific mortality in the SNC, accounting for the unlinked deaths and for the possible effect of change in death certificate coding was necessary.

A non-coding genomic duplication at the HMX1 locus is associated with crop ears in highland cattle.

Highland cattle with congenital crop ears have notches of variable size on the tips of both ears. In some cases, cartilage deformation can be seen and occasionally the external ears are shortened. We collected 40 cases and 80 controls across Switzerland. Pedigree data analysis confirmed a monogenic autosomal dominant mode of inheritance with variable expressivity. All affected animals could be traced back to a single common ancestor. A genome-wide association study was performed and the causative mutation was mapped to a 4 Mb interval on bovine chromosome 6. The H6 family homeobox 1 (HMX1) gene was selected as a positional and functional candidate gene. By whole genome re-sequencing of an affected Highland cattle, we detected 6 non-synonymous coding sequence variants and two variants in an ultra-conserved element at the HMX1 locus with respect to the reference genome. Of these 8 variants, only a non-coding 76 bp genomic duplication (g.106720058_106720133dup) located in the conserved region was perfectly associated with crop ears. The identified copy number variation probably results in HMX1 misregulation and possible gain-of-function. Our findings confirm the role of HMX1 during the development of the external ear. As it is sometimes difficult to phenotypically diagnose Highland cattle with slight ear notches, genetic testing can now be used to improve selection against this undesired trait.

Interaction between effects of genes coding for dopamine and glutamate transmission on striatal and parahippocampal function

The genes for the dopamine transporter (DAT) and the D-Amino acid oxidase activator (DAOA or G72) have been independently implicated in the risk for schizophrenia and in bipolar disorder and/or their related intermediate phenotypes. DAT and G72 respectively modulate central dopamine and glutamate transmission, the two systems most robustly implicated in these disorders. Contemporary studies have demonstrated that elevated dopamine function is associated with glutamatergic dysfunction in psychotic disorders. Using functional magnetic resonance imaging we examined whether there was an interaction between the effects of genes that influence dopamine and glutamate transmission (DAT and G72) on regional brain activation during verbal fluency, which is known to be abnormal in psychosis, in 80 healthy volunteers. Significant interactions between the effects of G72 and DAT polymorphisms on activation were evident in the striatum, parahippocampal gyrus, and supramarginal/angular gyri bilaterally, the right insula, in the right pre-/postcentral and the left posterior cingulate/retrosplenial gyri (P < 0.05, FDR-corrected across the whole brain). This provides evidence that interactions between the dopamine and the glutamate system, thought to be altered in psychosis, have an impact in executive processing which can be modulated by common genetic variation.

The long non-coding RNA NEAT1 is increased in IUGR placentas, leading to potential new hypotheses of IUGR origin/development.

INTRODUCTION Intrauterine Growth Restriction (IUGR) is a multifactorial disease defined by an inability of the fetus to reach its growth potential. IUGR not only increases the risk of neonatal mortality/morbidity, but also the risk of metabolic syndrome during adulthood. Certain placental proteins have been shown to be implicated in IUGR development, such as proteins from the GH/IGF axis and angiogenesis/apoptosis processes. METHODS Twelve patients with term IUGR pregnancy (birth weight < 10th percentile) and 12 CTRLs were included. mRNA was extracted from the fetal part of the placenta and submitted to a subtraction method (Clontech PCR-Select cDNA Subtraction). RESULTS One candidate gene identified was the long non-coding RNA NEAT1 (nuclear paraspeckle assembly transcript 1). NEAT1 is the core component of a subnuclear structure called paraspeckle. This structure is responsible for the retention of hyperedited mRNAs in the nucleus. Overall, NEAT1 mRNA expression was 4.14 (±1.16)-fold increased in IUGR vs. CTRL placentas (P = 0.009). NEAT1 was exclusively localized in the nuclei of the villous trophoblasts and was expressed in more nuclei and with greater intensity in IUGR placentas than in CTRLs. PSPC1, one of the three main proteins of the paraspeckle, co-localized with NEAT1 in the villous trophoblasts. The expression of NEAT1_2 mRNA, the long isoform of NEAT1, was only modestly increased in IUGR vs. CTRL placentas. DISCUSSION/CONCLUSION The increase in NEAT1 and its co-localization with PSPC1 suggests an increase in paraspeckles in IUGR villous trophoblasts. This could lead to an increased retention of important mRNAs in villous trophoblasts nuclei. Given that the villous trophoblasts are crucial for the barrier function of the placenta, this could in part explain placental dysfunction in idiopathic IUGR fetuses.