Regulation of MLH1 mRNA and protein expression by promoter methylation in primary colorectal cancer: a descriptive and prognostic cancer marker study.

BACKGROUND: In colorectal cancer MLH1 deficiency causes microsatellite instability, which is relevant for the patient's prognosis and treatment, and its putative heredity. Dysfunction of MLH1 is caused by sporadic gene promoter hypermethylation or by hereditary mutations as seen in Lynch Syndrome. The aim of this study was to determine in detail how DNA methylation regulates MLH1 expression and impacts clinical management. METHODS: Colorectal cancer samples were collected from 210 patients. The laboratory methods used to study these samples included methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), real-time quantitative PCR (qPCR), and immunohistochemistry (IHC). RESULTS: We found that the MLH1 mRNA and protein expression levels were highly related. MS-MLPA was successful in tumors from 195 patients. In these tumors, hypermethylation was observed in promoter regions A (n = 57), B (n = 30), C (n = 28), and D (n = 47), and in intron 1 (n = 25). The promoter region C and intron 1 methylation levels were found to be excellently suited for discriminating between low and high gene expression levels, whereas those of promoter regions A, B and D were less specific. Hypermethylation in any region (n = 77) served as an independent prognostic factor (hazard ratio 0.56, 95 % confidence interval 0.36-0.89, p = 0.01). CONCLUSIONS: MLH1 inactivation through hypermethylation was found to be related to improved survival. Hypermethylation in promoter region C and intron 1 served as the most specific markers for this inactivation.

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.

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.

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.

Conserved small non-coding RNAs that belong to the sigmaE regulon: role in down-regulation of outer membrane proteins.

Enteric bacteria respond to misfolded proteins by activating the transcription of "heat shock" genes. These genes are arranged in two major regulons controlled by the alternative sigma factors sigmaH and sigmaE. The two transcription factors coordinate the stress response in different cellular compartments; the sigmaH regulon is induced by stress in the cytoplasm whereas the sigmaE regulon is activated by stress signals in the cell envelope. In Escherichia coli sigmaE plays a central role in maintaining cell envelope integrity both under stress conditions and during normal growth. Previous work established that sigmaE is essential for viability of the bacterium and up-regulates expression of approximately 100 protein-encoding genes that influences nearly every aspect of the cell envelope. Moreover, the expression of several outer membrane proteins is down-regulated upon sigmaE activation. Here, we show that two Hfq-binding small RNAs, MicA and RybB, are under positive control of sigmaE. Transient induction of RybB resulted in decreased levels of the mRNAs encoding OmpC and OmpW. sigmaE -mediated regulation of ompC and ompW expression was abolished in strains lacking RybB or Hfq. Recently MicA was shown to act in destabilizing the ompA transcript when rapidly grown cells entered the stationary phase of growth. Also, the alternative sigma factor down-regulates this message in a small non-coding RNA-dependent fashion. These findings add the sigmaE regulon to the growing list of stress induced regulatory circuits that include small regulatory RNAs and provide insight in a homeostatic loop that prevent a build-up of unassembled outer membrane proteins in the envelope.

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.