Somatic and intergenerational G4C2 hexanucleotide repeat instability in a human C9orf72 knock-in mouse model.

Expansion of a G4C2 repeat in the C9orf72 gene is associated with familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). To investigate the underlying mechanisms of repeat instability, which occurs both somatically and intergenerationally, we created a novel mouse model of familial ALS/FTD that harbors 96 copies of G4C2 repeats at a humanized C9orf72 locus. In mouse embryonic stem cells, we observed two modes of repeat expansion. First, we noted minor increases in repeat length per expansion event, which was dependent on a mismatch repair pathway protein Msh2. Second, we found major increases in repeat length per event when a DNA double- or single-strand break (DSB/SSB) was artificially introduced proximal to the repeats, and which was dependent on the homology-directed repair (HDR) pathway. In mice, the first mode primarily drove somatic repeat expansion. Major changes in repeat length, including expansion, were observed when SSB was introduced in one-cell embryos, or intergenerationally without DSB/SSB introduction if G4C2 repeats exceeded 400 copies, although spontaneous HDR-mediated expansion has yet to be identified. These findings provide a novel strategy to model repeat expansion in a non-human genome and offer insights into the mechanism behind C9orf72 G4C2 repeat instability.

Human stanniocalcin-2 exhibits potent growth-suppressive properties in transgenic mice independently of growth hormone and IGFs.

Stanniocalcin (STC)-2 was discovered by its primary amino acid sequence identity to the hormone STC-1. The function of STC-2 has not been examined; thus we generated two lines of transgenic mice overexpressing human (h)STC-2 to gain insight into its potential functions through identification of overt phenotypes. Analysis of mouse Stc2 gene expression indicates that, unlike Stc1, it is not highly expressed during development but exhibits overlapping expression with Stc1 in adult mice, with heart and skeletal muscle exhibiting highest steady-state levels of Stc2 mRNA. Constitutive overexpression of hSTC-2 resulted in pre- and postnatal growth restriction as early as embryonic day 12.5, progressing such that mature hSTC-2-transgenic mice are approximately 45% smaller than wild-type littermates. hSTC-2 overexpression is sometimes lethal; we observed 26-34% neonatal morbidity without obvious dysmorphology. hSTC-2-induced growth retardation is associated with developmental delay, most notably cranial suture formation. Organ allometry studies show that hSTC-2-induced dwarfism is associated with testicular organomegaly and a significant reduction in skeletal muscle mass likely contributing to the dwarf phenotype. hSTC-2-transgenic mice are also hyperphagic, but this does not result in obesity. Serum Ca2+ and PO4 were unchanged in hSTC-2-transgenic mice, although STC-1 can regulate intra- and extracellular Ca2+ in mammals. Interestingly, severe growth retardation induced by hSTC-2 is not associated with a decrease in GH or IGF expression. Consequently, similar to STC-1, STC-2 can act as a potent growth inhibitor and reduce intramembranous and endochondral bone development and skeletal muscle growth, implying that these tissues are specific physiological targets of stanniocalcins.

Detection of lung cancer with volatile markers in the breath.

STUDY OBJECTIVES: To evaluate volatile organic compounds (VOCs) in the breath as tumor markers in lung cancer. Alkanes and monomethylated alkanes are oxidative stress products that are excreted in the breath, the catabolism of which may be accelerated by polymorphic cytochrome p450-mixed oxidase enzymes that are induced in patients with lung cancer. DESIGN: Combined case-control and cross-sectional study. SETTING: Five academic pulmonary medicine services in the United States and the United Kingdom. PATIENTS AND PARTICIPANTS: One hundred seventy-eight bronchoscopy patients and 41 healthy volunteers. INTERVENTION: Breath samples were analyzed by gas chromatography and mass spectroscopy to determine alveolar gradients (ie, the abundance in breath minus the abundance in room air) of C4-C20 alkanes and monomethylated alkanes. MEASUREMENTS: Patients with primary lung cancer (PLC) were compared to healthy volunteers, and a predictive model was constructed using forward stepwise discriminant analysis of the alveolar gradients. This model was cross-validated with a leave-one-out jackknife technique and was tested in two additional groups of patients who had not been used to develop the model (ie, bronchoscopy patients in whom cancer was not detected, and patients with metastatic lung cancer [MLC]). RESULTS: Eighty-seven of 178 patients had lung cancer (PLC, 67 patients; MLC, 15 patients; undetermined, 5 patients). A predictive model employing nine VOCs identified PLC with a sensitivity of 89.6% (60 of 67 patients) and a specificity of 82.9% (34 of 41 patients). On cross-validation, the sensitivity was 85.1% (57 of 67 patients) and the specificity was 80.5% (33 of 41 patients). The stratification of patients by tobacco smoking status, histologic type of cancer, and TNM stage of cancer revealed no marked effects. In the two additional tests, the model predicted MLC with a sensitivity of 66.7% (10 of 15 patients), and it classified the cancer-negative bronchoscopy patients with a specificity of 37.4% (34 of 91 patients). CONCLUSIONS: Compared to healthy volunteers, patients with PLC had abnormal breath test findings that were consistent with the accelerated catabolism of alkanes and monomethylated alkanes. A predictive model employing nine of these VOCs exhibited sufficient sensitivity and specificity to be considered as a screen for lung cancer in a high-risk population such as adult smokers.

Overexpression of human stanniocalcin affects growth and reproduction in transgenic mice.

In mammals stanniocalcin (STC) is widely expressed, and in the kidney and gut it regulates serum calcium levels by promoting phosphate reabsorption. To shed further light on its functional significance in mammals we have created several lines of mice that express a human STC (hSTC) transgene. Three lines expressed the hSTC transgene, but only two lines exhibited high expression and contained circulating hSTC, and in these animals there was a reduction in postnatal growth (30-50%) that persisted after weaning. Moreover, even wild-type pups exhibited a growth retardation phenotype when nursed by a transgenic foster mother, and this implies that hSTC overexpression deleteriously affects maternal behavior and/or lactation. The reproductive potential of female transgenic mice was also compromised, as evidenced by significantly smaller litter sizes, but transgenic male fertility was unchanged even though the transgene was most highly expressed in testes. Interestingly, transgene-derived serum hSTC increased significantly after puberty and was severalfold higher in females than in males, suggesting a gender-specific mechanism for maintaining elevated circulating levels of STC. Blood analysis revealed that both transgenic lines had elevated phosphate and decreased alkaline phosphatase levels, indicative of altered kidney and bone metabolism. These studies provide the first evidence that STC is involved in growth and reproduction and reaffirm its role in mineral homeostasis.