Anders Aamann Rasmussen

Regulation of ompA mRNA stability: the role of a small regulatory RNA in growth phase‐dependent control

The Escherichia coli ompA mRNA, encoding a highly abundant outer membrane protein, has served as a model for regulated mRNA decay in bacteria. The half‐life of this transcript correlates inversely with the bacterial growth rate and is growth stage‐dependent. The stability of the messenger is determined by the 5′‐untranslated region which possesses cleavage sites for RNase E. Hfq binds to this region, is essential for controlling the stability and has been suggested to directly regulate ompA mRNA decay. Here we report that the 78 nucleotide SraD RNA, which is highly conserved among Enterobacteriaceae, acts in destabilizing the ompA transcript when rapidly grown cells enter the stationary phase of growth. During this growth‐stage the expression of SraD RNA becomes strongly increased. The SraD‐mediated decay of ompA mRNA depends on Hfq and in vitro studies revealed that Hfq facilitates binding of the regulatory RNA to the translational initiation region of the messenger. Deletion of sraD, however, does not significantly affect the stability of the ompA mRNA in slowly growing cells. Our results indicate that distinct regulatory circuits are responsible for growth phase‐ and growth rate‐dependent control of the ompA mRNA stability.

A conserved small RNA promotes silencing of the outer membrane protein YbfM

In the past few years an increasing number of small non‐coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the post‐transcriptional level. These RNAs act under various growth and stress conditions, suggesting that one important physiological role of regulatory RNA molecules in Gram‐negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP–sRNA network by showing that the expression of the OMP YbfM is silenced by a conserved sRNA, designated MicM (also known as RybC/SroB). The regulation is strictly dependent on the RNA chaperone Hfq, and mutational analysis indicates that MicM sequesters the ribosome binding site of ybfM mRNA by an antisense mechanism. Furthermore, we provide evidence that Hfq strongly enhances the on‐rate of duplex formation between MicM and its target RNA in vitro, supporting the idea that a major cellular role of the RNA chaperone is to act as a catalyst in RNA–RNA duplex formation.

Heart defects and other features of the 22q11 distal deletion syndrome.

22q11.2 distal deletion syndrome is distinct from the common 22q11.2 deletion syndrome and caused by microdeletions localized adjacent to the common 22q11 deletion at its telomeric end. Most distal deletions of 22q11 extend from LCR22-4 to an LCR in the range LCR22-5 to LCR22-8. We present three patients with 22q11 distal deletions, of whom two have complex congenital heart malformation, thus broadening the phenotypic spectrum. We compare cardiac malformations reported in 22q11 distal deletion to those reported in the common 22q11 deletion syndrome. We also review the literature for patients with 22q11 distal deletions, and discuss the possible roles of haploinsufficiency of the MAPK1 gene. We find the most frequent features in 22q11 distal deletion to be developmental delay or learning disability, short stature, microcephalus, premature birth with low birth weight, and congenital heart malformation ranging from minor anomalies to complex malformations. Behavioral problems are also seen in a substantial portion of patients. The following dysmorphic features are relatively common: smooth philtrum, abnormally structured ears, cleft palate/bifid uvula, micro-/retrognathia, upslanting palpebral fissures, thin upper lip, and ear tags. Very distal deletions including region LCR22-6 to LCR22-7 encompassing the SMARCB1-gene are associated with an increased risk of malignant rhabdoid tumors.

Identification of BRCA1-deficient ovarian cancers.

Objective. It is believed that 24–40% of ovarian cancers have dysfunction in the BRCA1 or BRCA2 (BRCAness) genes, due to either inherited or somatic mutations or due to epigenetic inactivation. Demonstration of ovarian cancers with BRCAness is becoming important both due to the possibility of offering genetic counseling and due to beneficial effects of polyadenosine diphosphate ribose polymerase inhibitor treatment in this group. As DNA sequencing is expensive and time consuming, efforts have been devoted to develop more indirect methods for BRCA screening that can improve the selection of patients for sequence‐based BRCA testing. Design. BRCA1 immunohistochemistry, fluorescence in situ hybridization (FISH) and methylation analyses were performed on formalin‐fixed, paraffin‐embedded ovarian cancer tissue. Sample. Fifty‐four ovarian cancers; 15 BRCA1 cancers, four BRCA2 cancers, 10 cancers from patients with a family history but no mutation detected, and 25 ovarian cancers with unknown BRCA1 status. Results. Abnormal BRCA1 immunohistochemistry was found to indicate BRCA mutations with a sensitivity of 80%, a specificity of 93% and an estimated positive predictive value of 73%. The FISH analyses supported the diagnosis in most cases. Methylation analyses could indicate BRCA deficiency in combination with one of the other methods. Conclusions. BRCA1 immunohistochemistry is a promising screening method for BRCA1 mutation detection.

Triploid pregnancies: genetic and clinical features of 158 cases.

OBJECTIVE The purpose of this study was to analyze the correlation between the genetic constitution and the phenotype in triploid pregnancies. STUDY DESIGN One hundred fifty-eight triploid pregnancies were identified in hospitals in Western Denmark from April 1986 to April 2010. Clinical data and karyotypes were collected retrospectively, and archived samples were retrieved. The parental origin of the genome, either double paternal contribution (PPM) or double maternal contribution (MMP) was determined by an analysis of methylation levels at imprinted sites. RESULTS There were significantly more PPM than MMP cases (P < .01). In MMP cases, the possible karyotypes had similar frequencies, whereas, in PPM cases, 43% had the karyotype 69,XXX, 51% had the karyotype 69,XXY, and 6% had the karyotype 69,XYY. Molar phenotype was seen only in PPM cases. However, PPM cases with a nonmolar phenotype were also seen. For both parental genotypes, various fetal phenotypes were seen at autopsy. Levels of human chorionic gonadotropin in maternal serum were low in MMP cases and varying in PPM cases, some being as low as in the MMP cases. CONCLUSION In a triploid pregnancy, suspicion of hydatidiform mole at ultrasound scanning, by macroscopic inspection of the evacuated tissue, at histology, or because of a high human chorionic gonadotropin in maternal serum level each predict the parental type PPM with a very high specificity. In contrast, the sensitivity of these observations was <100%.

Methylation-specific multiplex ligation-dependent probe amplification: utility for prenatal diagnosis of parental origin in human triploidy

When a triploid pregnancy is diagnosed prenatally, gynaecologists have traditionally relied on the histopathological examination of the tissue from the terminated pregnancy to determine if the pregnancy is molar. However, reproducibility is poor and variability is high when diagnosing hydatidiform moles. Triploid pregnancies can have either the chromosomal constitution of two maternal and one paternal set, or two paternal and one maternal set, but only the conceptuses with two paternal sets have the potential to cause maternal complications. Therefore, it would be beneficial to introduce a method that gives the gynaecologist the parental origin of the genome of the triploid conceptus as early as possible, without delaying the process by first collecting parental samples.

Thermodynamic stability and denaturation kinetics of a benign natural transthyretin mutant identified in a Danish kindred

The disease phenotype of transthyretin (TTR) is dramatically influenced by single point mutations in the TTR gene. Herein, we report on a novel mutation D99N (Asp99Asn) in TTR found in a Danish kindred. None of the family members carrying this mutation have so far shown any clinical signs of amyloidosis. One carrier found compound heterozygous for TTR D99N and L111M (Leu111Met) associated with cardiac amyloid is asymptomatic (42 years). Disease severity can often be linked to both the kinetics of fibril formation and the degree of destabilisation of the native state. In this study, we show that the thermodynamic stability and rate of tetramer dissociation of the variant TTR D99N is unchanged or slightly more stable than wild type (WT) TTR. Furthermore, the in vitro fibrillation kinetics of the variant reveals an unchanged or slightly suppressed tendency to form fibrils compared to WT. Thus, the in vitro experiments support the lack of clinical symptoms observed so far for the TTR D99N carriers. In line with this, studies on kinetic stability and fibrillation kinetics reveal indistinguishable stability of TTR heterotetramers D99N/L111M compared to the heterotetramers WT/L111M. In conclusion, TTR D99N is predicted to be a non-pathogenic benign mutation with WT properties.

Are mutations in K-RAS, BRAF and PIK3CA genes critical for response to adjuvant trastuzumab treatment in patients with HER-2 positive breast cancer?

Abstract Background: HER-2 is a prognostic and predictive factor in patients suffering from breast cancer. Since 2006 we have tested routinely the HER-2 status in patients with primary breast cancer at Vejle Hospital,