Mutation analysis in Bardet-Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals

Bardet–Biedl syndrome (BBS) is a rare, primarily autosomal-recessive ciliopathy. The phenotype of this pleiotropic disease includes retinitis pigmentosa, postaxial polydactyly, truncal obesity, learning disabilities, hypogonadism and renal anomalies, among others. To date, mutations in 15 genes (BBS1–BBS14, SDCCAG8) have been described to cause BBS. The broad genetic locus heterogeneity renders mutation screening time-consuming and expensive. We applied a strategy of DNA pooling and subsequent massively parallel resequencing (MPR) to screen individuals affected with BBS from 105 families for mutations in 12 known BBS genes. DNA was pooled in 5 pools of 21 individuals each. All 132 coding exons of BBS1–BBS12 were amplified by conventional PCR. Subsequent MPR was performed on an Illumina Genome Analyzer II(™) platform. Following mutation identification, the mutation carrier was assigned by CEL I endonuclease heteroduplex screening and confirmed by Sanger sequencing. In 29 out of 105 individuals (28%), both mutated alleles were identified in 10 different BBS genes. A total of 35 different disease-causing mutations were confirmed, of which 18 mutations were novel. In 12 additional families, a total of 12 different single heterozygous changes of uncertain pathogenicity were found. Thus, DNA pooling combined with MPR offers a valuable strategy for mutation analysis of large patient cohorts, especially in genetically heterogeneous diseases such as BBS

Whole-exome resequencing distinguishes cystic kidney diseases from phenocopies in renal ciliopathies

Rare single-gene disorders cause chronic disease. However, half of the 6,000 recessive single gene causes of disease are still unknown. Because recessive disease genes can illuminate, at least in part, disease pathomechanism, their identification offers direct opportunities for improved clinical management and potentially treatment. Rare diseases comprise the majority of chronic kidney disease (CKD) in children but are notoriously difficult to diagnose. Whole exome resequencing facilitates identification of recessive disease genes. However, its utility is impeded by the large number of genetic variants detected. We here overcome this limitation by combining homozygosity mapping with whole exome resequencing in 10 sib pairs with a nephronophthisis-related ciliopathy, which represents the most frequent genetic cause of CKD in the first three decades of life. In 7 of 10 sib-ships with a histologic or ultrasonographic diagnosis of nephronophthisis-related ciliopathy we detect the causative gene. In six sib-ships we identify mutations of known nephronophthisis-related ciliopathy genes, while in two additional sib-ships we found mutations in the known CKD-causing genes SLC4A1 and AGXT as phenocopies of nephronophthisis-related ciliopathy. Thus whole exome resequencing establishes an efficient, non-invasive approach towards early detection and causation-based diagnosis of rare kidney diseases. This approach can be extended to other rare recessive disorders, thereby providing accurate diagnosis and facilitating the study of disease mechanisms

Twelve-Day Reinforcement-Based Memory Retention in African Cichlids (Labidochromis caeruleus)

The formation of long-term memories for food sources is essential for the survival of most animals. Long-term memory formation in mammalian species has been demonstrated through a variety of conditioning tasks, however, the nature of long-term memory in fish is less known. In the current study, we explored whether African cichlids (Labidochromis caeruleus) could form memories for food-reinforced stimuli that last for 12 days. During the training sessions, fish were reinforced for approaching an upward drifting line grating. After a rest period of 12 days, fish demonstrated a significant preference for the upward drifting grating. To determine whether this preference could also be reversed, fish were then reinforced for approaching a downward drifting line grating after a 20-day rest period. When tested 12 days later, there were no significant differences in preference for either stimulus; however, following a second training period for the downward stimulus, there was a significant preference for the downward drifting grating. This suggests that cichlids are able to form reversible discrimination-based memories for food-reinforced stimuli that remain consolidated for at least 12 days

Evaluation of the first-in-class antimitochondrial metabolism agent CPI-613 in hematologic malignancies.

6524^ Background: Altered metabolism is a hallmark of cancer, including hematologic malignancies. Most tumor cells use glycolysis even under aerobic conditions (the Warburg effect). This altered metabolism is a possible therapeutic target. The novel lipoate derivative CPI-613 is a first-in-class agent that targets a key mitochondrial enzyme, pyruvate dehydrogenase complex (PDH). Methods: CPI-613 was tested against leukemia cell lines in vitro and in vivo. It is also the subject of a phase I clinical trial for patients with hematologic malignancies. Results: CPI-613 was active against HL60, Jurkat, and K562 cells, with an average IC50 value of 14 μM (range 12.2 – 16.4). CPI-613 was synergistic with doxorubicin, with Combinatorial Index (CI) values of 0.478 to 0.765. CPI-613 sensitivity increased with knockdown of p53 or MN1 expression, despite their increased resistance to standard therapy. CPI-613 was synergistic with nilotinib against BCR-ABL-expressing cells and with sorafenib against Flt3 ITD-expressing cells. CI values for nilotinib + CPI was 0.059 (+/-0.002) and for sorafenib + CPI was 0.581 (+/-0.052). In vivo, CPI-613 synergized with doxorubicin; median survival improved from 12 days with doxorubicin to 16 days with CPI-613 plus doxorubicin (p=0.0001). In the phase I clinical trial, 7 of 13 evaluable patients had a response of stable disease or better, for an overall response rate of 54% (see table). CPI-613 was well tolerated, with no evidence of marrow suppression and no dose limiting toxicity identified. Conclusions: The novel agent CPI-613 has activity against a variety of hematological malignancies, including acute leukemias. The therapeutic index appears high, with only minor toxicities observed. [Table: see text] </jats:p

Older adults’ experiences of group-based physical activity : A qualitative study from the ‘GOAL’ randomized controlled trial

Objectives: In this qualitative study, we examined older adults’ experiences of taking part in two 2 efficacious group-based physical activity programs as part of the GrOup-based physical Activity for 3 oLder adults (GOAL) randomized controlled trial. 4 Method: In the GOAL Trial, 627 older adults were randomized to one of three conditions: similar 5 age same gender (SASG), similar age mixed gender (SAMG), or ‘standard’ mixed age mixed 6 gender (MAMG) control exercise group conditions. Participants in this qualitative study (N = 31; 7 Nmen = 17, Nwomen = 14; Mage = 70 years) were purposively sampled from the two experimental 8 conditions (SASG, SAMG) and involved in semi-structured interviews. The data were analyzed 9 using thematic analysis. 10 Results: The results included 12 lower-order and three higher-order themes that reflected (a) the 11 benefits and challenges of exercising with peers, (b) the group as a means of mitigating social 12 isolation, and (c) group exercise and the physically active body. 13 Discussion: Findings highlight the importance of social connections that exist within the age-14 matched physical activity programs, as well as some of the challenges for older adults participating 15 in community-based physical activity programs. Implications for intervention, program planning, 16 and future research are discussed.Education, Faculty ofNon UBCKinesiology, School ofUnreviewedFacult

PDK1 Attenuation Fails to Prevent Tumor Formation in PTEN-Deficient Transgenic Mouse Models

Abstract PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target. Cancer Res; 71(8); 3052–65. ©2011 AACR.</jats:p