The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3’ splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.     

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 µm tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells – on the level of a few protein molecules in germinal centers.     

Haemolytic anaemia in Basenji dogs. 2. Partial deficiency of erythrocyte pyruvate kinase (PK; EC 2.7.1.40) in heterozygous carriers

Congenital nonspherocytic haemolytic anaemia (HA) in dogs of the Basenji breed is inherited as a simple, autosomal recessive trait. Previous results of pyruvate kinase (PK) assays suggest a causal relationship between the anaemia and PK deficiency in erythrocytes. In the present investigation assays of this enzyme have been performed on haemolysates from 45 Basenji dogs, comprising 3 anaemic and 42 non-anaemic individuals of which 13 were known heterozygotes. The PK activity in haemolysates from the 42 non-anaemic dogs exhibited a bimodal distribution corresponding to the genotypic classes: heterozygotes and normal homozygotes. The results indicate that heterozygotes have a partial, detectable enzyme deficiency, not reflected in clinical disease, and thus give evidence of a gene dosage effect in agreement with observations in man. The proposed genotypes PK PK, PK pk and pk pk refer to normal homozygotes, heterozygotes, and anaemic individuals, respectively. The findings strengthen the genetic hypothesis of recessiveness of the anaemia by direct detection of heterozygosity of parents of affected individuals. Moreover, the results are of value in comparative studies and they have practical application in connection with eradication programmes.     

Bayesian Spatio-Temporal Analysis and Geospatial Risk Factors of Human Monocytic Ehrlichiosis

Variations in spatio-temporal patterns of Human Monocytic Ehrlichiosis (HME) infection in the state of Kansas, USA were examined and the relationship between HME relative risk and various environmental, climatic and socio-economic variables were evaluated. HME data used in the study was reported to the Kansas Department of Health and Environment between years 2005–2012, and geospatial variables representing the physical environment [National Land cover/Land use, NASA Moderate Resolution Imaging Spectroradiometer (MODIS)], climate [NASA MODIS, Prediction of Worldwide Renewable Energy (POWER)], and socio-economic conditions (US Census Bureau) were derived from publicly available sources. Following univariate screening of candidate variables using logistic regressions, two Bayesian hierarchical models were fit; a partial spatio-temporal model with random effects and a spatio-temporal interaction term, and a second model that included additional covariate terms. The best fitting model revealed that spatio-temporal autocorrelation in Kansas increased steadily from 2005–2012, and identified poverty status, relative humidity, and an interactive factor, ‘diurnal temperature range x mixed forest area’ as significant county-level risk factors for HME. The identification of significant spatio-temporal pattern and new risk factors are important in the context of HME prevention, for future research in the areas of ecology and evolution of HME, and as well as climate change impacts on tick-borne diseases.     

Structural organization of the human short-chain acyl-CoA dehydrogenase gene

Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme that catalyzes the initial reaction in short-chain fatty acid β-oxidation. Defects in the SCAD enzyme are associated with failure to thrive, often with neuromuscular dysfunction and elevated urinary excretion of ethylmalonic acid (EMA). To define the genetic basis of SCAD deficiency and ethylmalonic aciduria in patients, we have determined the sequence of the complete coding portion of the human SCAD gene (ACADS) and all of the intron-exon boundaries. The SCAD gene is approximately 13 kb in length and consists of 10 exons. Four polymorphic sites have previously been detected by sequencing of cDNA from fibroblasts of patients excreting elevated amounts of EMA. Three of these polymorphisms (321T/C, 990C/T, 1260G/C) are silent variants, while a 625G/A polymorphism results in an amino acid replacement and has been shown to be associated with ethylmalonic aciduria. From analysis of 18 unrelated Danish families, we show that the four SCAD gene polymorphisms constitute five allelic variants of the SCAD gene, and that the 625A variant together with the less frequent variant form of the three other polymorphisms (321C, 990T, 1260C) constitutes an allelic variant with a frequency of 22% in the general Danish population. Using fluorescence in-situ hybridization, we confirm the localization of the human SCAD gene to the distal part of Chromosome (Chr) 12 and suggest that the SCAD gene is a single-copy gene. The evolutionary relationship between SCAD and five other members of the acyl-CoA dehydrogenase family was investigated by two independent approaches that gave similar phylogenetic trees. Received: 10 April 1997 / Accepted: 8 August 1997