Correction to the pathogenic alternative splicing, caused by the common GNB3 c.825C>T allele, using a novel, antisense morpholino

The very common GNB3 c.825C>T polymorphism (rs5443), is present in approximately half of all human chromosomes. Significantly the presence of the GNB3 825T allele has been strongly associated, with predisposition to essential hypertension. Paradoxically the presence of the GNB3 825T allele, in exon 10, introduces a pathogenic alternative RNA splice site into the middle of exon 9. To attempt to correct this pathogenic aberrant splicing, we therefore bioinformatically designed, using a Gene Tools® algorithm, a GNB3 specific, antisense morpholino. It was hoped that this morpholino would behave in vitro as either a potential “ splice blocker and/or exon skipper, to both bind and inhibit/reduce the aberrant splicing of the GNB3, 825T allele. On transfecting a human lymphoblast cell line homozygous for the 825T allele, with this antisense morpholino, we encouragingly observed both a significant reduction (from ~58% to ~5%) in the production of the aberrant smaller GNB3 transcript, and a subsequent increase in the normal GNB3 transcript (from ~42% to ~95%). Our results demonstrate the potential use of a GNB3 specific antisense morpholino, as a pharmacogenetic therapy for essential hypertension

Mutation in the guanine nucleotide-binding protein beta-3 causes retinal degeneration and embryonic mortality in chickens

PURPOSE. To identify the gene defect that causes blindness and the predisposition to embryonic death in the retinopathy globe enlarged (rge) chicken. METHODS. Linkage analysis, with previously uncharacterized microsatellite markers from chicken chromosome 1, was performed on 138 progeny of an rge/+ and an rge/rge cross, and candidate genes were sequenced. RESULTS. The rge locus was refined and the gene for guanine nucleotide-binding protein β-3 (GNB3), which encodes a cone transducin β subunit, was found to have a 3-bp deletion (D153del) that segregated with the rge phenotype. This mutation deleted a highly conserved aspartic acid residue in the third of seven WD domains in GNB3. In silico modeling suggested that this mutation destabilized the protein. Furthermore, a 70% reduction was found in immunoreactivity to anti-GNB3 in the rge-affected retina. CONCLUSIONS. These findings implicate the β-subunit of cone transducin as the defective protein underlying the rge phenotype. Furthermore, GNB3 is ubiquitously expressed, and the c.825C→T GNB3 splicing variant (MIM 139130) has been associated with hypertension, obesity, diabetes, low birth weight, coronary heart disease, and stroke in the human population. It therefore seems likely that the defect underlying these human diseases also causes reduced embryonic viability in the rge chicken, making it a powerful model for studying the pathology involved in these associations

The alternate GNB3 splice variant, Gβ3s, exhibits an altered signalling response to EGF stimulation, which leads to enhanced cell migration

It has recently been reported that the duplication of the GNB3 gene has been shown to be directly linked to an obesity phenotype, both in humans and also in a humanised mouse model. Moreover, the common human GNB3 c.825C>T polymorphism (rs5443) causes this ubiquitously expressed gene to be aberrantly spliced approximately 50% of the time leading to the production of both a normal Gβ3 protein and a truncated, possibly less stable subunit, known as Gβ3s. The presence of the GNB3 825T allele has previously been shown to be associated with predisposition to hypertension, obesity, various cancers, Alzheimers, age related cognitive function, erectile dysfunction as well as a marker for pharmacogenetic drug action. Great controversy, however, currently exists as to whether these phenotypes associated with the 825T allele are a) mainly due to the presence of the smaller, possibly more active, Gβ3s subunit or b) merely down to the haploinsufficiency of the normal GNB3 transcript, due to its frequent aberrant splicing. In order to try and address these two conflicting hypothesis, we report on the identification and characterisation of signalling alterations unique to the presence of Gβ3s protein subunit. Moreover we also show the physiological consequences associated with altered signalling, directly induced by the Gβ3s subunit. For this, we used both an EBV transformed lymphoblast cell line homozygote for GNB3 825T/825T (TT) and a stable Gβ3s expressing recombinant COS-7 clone. In both of these cell lines that express the Gβ3s subunit, we found enhanced cytosolic calcium influx upon stimulation with EGF, TGFα and VEGF ligands, as compared to “normal” GNB3 controls with the 825C/825C (CC) genotype. This aberrant calcium influx also led to an increase in ERK, but not AKT1, phosphorylation. Despite the lack of AKT1 activation, we paradoxically observed a significant increase in phosphorylation of its downstream substrates, namely mTOR and p70S6k (KS6B2). Moreover we observed a decrease in phospho FoxO3a only in Gβ3s expressing cells, but not in the “normal” GNB3 (CC) control cell line. The presence of the Gβ3s subunit also appeared to alter the distinct localisation patterns of both Foxo3a and AKT1, while also increasing the colocalisation of mTOR and p70S6K. Subsequent growth factor stimulation studies revealed that EGF treatment, of Gβ3s expressing cells, appeared to cause a significant decrease in cAMP levels, which, in turn resulted in both enhanced caveolin-1a phosphorylation, and an increase in actin stress fibre formation. The identification of these distinct Gβ3s specific signalling alterations were indicative of a more aggressive migratory phenotype. This led us to further investigate and confirm that the presence of the Gβ3s subunit also appears to cause significantly enhanced migration and robust scratch wound healing kinetics, as compared to cells harbouring only the normal copy of the gene. These data therefore present convincing evidence that the Gβ3s subunit is stable, functional and its presence can significantly alter signalling pathways, in different cell types

New measurement paradigms

This collection of New Measurement Paradigms papers represents a snapshot of the variety of measurement methods in use at the time of writing across several projects funded by the National Science Foundation (US) through its REESE and DR K–12 programs. All of the projects are developing and testing intelligent learning environments that seek to carefully measure and promote student learning, and the purpose of this collection of papers is to describe and illustrate the use of several measurement methods employed to achieve this. The papers are deliberately short because they are designed to introduce the methods in use and not to be a textbook chapter on each method. The New Measurement Paradigms collection is designed to serve as a reference point for researchers who are working in projects that are creating e-learning environments in which there is a need to make judgments about students’ levels of knowledge and skills, or for those interested in this but who have not yet delved into these methods

BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers

Background: The K3326X variant in BRCA2 (BRCA2*c.9976A>T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers. Methods: Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided. Results: The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10- 6) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10-3). These associations were stronger for serous ovarian cancer and for estrogen receptor–negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10-5 and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10-5, respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed. Conclusions: Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations

Common genetic variation in cellular transport genes and epithelial ovarian cancer (EOC) risk

Background Defective cellular transport processes can lead to aberrant accumulation of trace elements, iron, small molecules and hormones in the cell, which in turn may promote the formation of reactive oxygen species, promoting DNA damage and aberrant expression of key regulatory cancer genes. As DNA damage and uncontrolled proliferation are hallmarks of cancer, including epithelial ovarian cancer (EOC), we hypothesized that inherited variation in the cellular transport genes contributes to EOC risk. Methods In total, DNA samples were obtained from 14,525 case subjects with invasive EOC and from 23,447 controls from 43 sites in the Ovarian Cancer Association Consortium (OCAC). Two hundred seventy nine SNPs, representing 131 genes, were genotyped using an Illumina Infinium iSelect BeadChip as part of the Collaborative Oncological Gene-environment Study (COGS). SNP analyses were conducted using unconditional logistic regression under a log-additive model, and the FDR q<0.2 was applied to adjust for multiple comparisons. Results The most significant evidence of an association for all invasive cancers combined and for the serous subtype was observed for SNP rs17216603 in the iron transporter gene HEPH (invasive: OR = 0.85, P = 0.00026; serous: OR = 0.81, P = 0.00020); this SNP was also associated with the borderline/low malignant potential (LMP) tumors (P = 0.021). Other genes significantly associated with EOC histological subtypes (p<0.05) included the UGT1A (endometrioid), SLC25A45 (mucinous), SLC39A11 (low malignant potential), and SERPINA7 (clear cell carcinoma). In addition, 1785 SNPs in six genes (HEPH, MGST1, SERPINA, SLC25A45, SLC39A11 and UGT1A) were imputed from the 1000 Genomes Project and examined for association with INV EOC in white-European subjects. The most significant imputed SNP was rs117729793 in SLC39A11 (per allele, OR = 2.55, 95% CI = 1.5-4.35, p = 5.66x10-4). Conclusion These results, generated on a large cohort of women, revealed associations between inherited cellular transport gene variants and risk of EOC histologic subtypes

LHC String Phenomenology

We argue that it is possible to address the deeper LHC Inverse Problem, to gain insight into the underlying theory from LHC signatures of new physics. We propose a technique which may allow us to distinguish among, and favor or disfavor, various classes of underlying theoretical constructions using (assumed) new physics signals at the LHC. We think that this can be done with limited data $(5-10 fb^{-1})$, and improved with more data. This is because of two reasons -- a) it is possible in many cases to reliably go from (semi)realistic microscopic string construction to the space of experimental observables, say, LHC signatures. b) The patterns of signatures at the LHC are sensitive to the structure of the underlying theoretical constructions. We illustrate our approach by analyzing two promising classes of string compactifications along with six other string-motivated constructions. Even though these constructions are not complete, they illustrate the point we want to emphasize. We think that using this technique effectively over time can eventually help us to meaningfully connect experimental data to microscopic theory.Comment: 50 Pages, 13 Figures, 3 Tables, v2: minor changes, references adde

Cervical lymph node metastasis in high-grade transformation of head and neck adenoid cystic carcinoma: a collective international review

Adenoid cystic carcinoma (AdCC) is among the most common malignant tumors of the salivary glands. It is characterized by a prolonged clinical course, with frequent local recurrences, late onset of metastases and fatal outcome. High-grade transformation (HGT) is an uncommon phenomenon among salivary carcinomas and is associated with increased tumor aggressiveness. In AdCC with high-grade transformation (AdCC-HGT), the clinical course deviates from the natural history of AdCC. It tends to be accelerated, with a high propensity for lymph node metastasis. In order to shed light on this rare event and, in particular, on treatment implications, we undertook this review: searching for all published cases of AdCC-HGT. We conclude that it is mandatory to perform elective neck dissection in patients with AdCC-HGT, due to the high risk of lymph node metastases associated with transformation

Homozygous in-frame deletion in CATSPERE in a man producing spermatozoa with loss of CatSper function and compromised fertilizing capacity

Study question: Does a man (patient 1) with a previously described deficiency in principle cation channel of sperm (CatSper) function have a mutation in the CatSper-epsilon (CATSPERE) and/or CatSper-zeta (CATSPERZ) gene?Summary answer: Patient 1 has a homozygous in-frame 6-bp deletion in exon 18 (c.2393-2398delCTATGG, rs761237686) of CATSPERE. WHAT IS KNOWN ALREADY: CatSper is the principal calcium channel of mammalian spermatozoa. Spermatozoa from patient 1 had a specific loss of CatSper function and were unable to fertilize at IVF. Loss of CatSper function could not be attributed to genetic abnormalities in coding regions of seven CatSper subunits. Two additional subunits (CatSper-epsilon (CATPSERE) and CatSper-zeta (CATSPERZ)) were recently identified, and are now proposed to contribute to the formation of the mature channel complex. STUDY DESIGN, SIZE, DURATION: This was a basic medical research study analysing genomic data from a single patient (patient 1) for defects in CATSPERE and CATSPERZ.Participants/materials, setting, methods: The original exome sequencing data for patient 1 were analysed for mutations in CATSPERE and CATSPERZ. Sanger sequencing was conducted to confirm the presence of a rare variant.Main results and the role of chance: Patient 1 is homozygous for an in-frame 6-bp deletion in exon 18 (c.2393-2398delCTATGG, rs761237686) of CATSPERE that is predicted to be highly deleterious.Limitations, reasons for caution: The nature of the molecular deficit caused by the rs761237686 variant and whether it is exclusively responsible for the loss of CatSper function remain to be elucidated.Wider implications of the findings: Population genetics are available for a significant number of predicted deleterious variants of CatSper subunits. The consequence of homozygous and compound heterozygous forms on sperm fertilization potential could be significant. Selective targeting of CatSper subunit expression maybe a feasible strategy for the development of novel contraceptives.</p