Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy

Background Nephronophthisis associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity, a strategy of DNA pooling with consecutive massively parallel resequencing (MPR) was devised.Methods In 120 patients with severe NPHP-AC phenotypes, five pools of genomic DNA with 24 patients each were prepared which were used as templates in order to PCR amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on an Illumina Genome-Analyser and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease based heteroduplex screening and confirmed by Sanger sequencing.Results For proof of principle, DNA from patients with known mutations was used and detection of 22 out of 24 different alleles (92% sensitivity) was demonstrated. MPR led to the molecular diagnosis in 30/120 patients (25%) and 54 pathogenic mutations (27 novel) were identified in seven different NPHP-AC genes. Additionally, in 24 patients only single heterozygous variants of unknown significance were found.Conclusions The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single gene disorders. The lack of mutations in 75% of patients in this cohort indicates further extensive heterogeneity in NPHP-AC

Biophysical interactions in tropical agroforestry systems

sequential systems, simultaneous systems Abstract. The rate and extent to which biophysical resources are captured and utilized by the components of an agroforestry system are determined by the nature and intensity of interac-tions between the components. The net effect of these interactions is often determined by the influence of the tree component on the other component(s) and/or on the overall system, and is expressed in terms of such quantifiable responses as soil fertility changes, microclimate modification, resource (water, nutrients, and light) availability and utilization, pest and disease incidence, and allelopathy. The paper reviews such manifestations of biophysical interactions in major simultaneous (e.g., hedgerow intercropping and trees on croplands) and sequential (e.g., planted tree fallows) agroforestry systems. In hedgerow intercropping (HI), the hedge/crop interactions are dominated by soil fertility improvement and competition for growth resources. Higher crop yields in HI than in sole cropping are noted mostly in inherently fertile soils in humid and subhumid tropics, and are caused by large fertility improvement relative to the effects of competition. But, yield increases are rare in semiarid tropics and infertile acid soils because fertility improvement does not offse

Effects of insulin-like growth factor binding proteins in bone - a matter of cell and site

The actions of the insulin-like growth factor (IGF)-system are controlled by six IGF-binding proteins (IGFBPs). The IGFBPs are thought to affect local effects of IGF-I and IGF-II due to higher affinity if compared to IGF-I receptors and due to cell-type specific IGFBP expression patterns. It was found in IGFBP knockout models that the IGFBP family is functionally redundant. Thus, functional analysis of potential effects of IGFBPs is dependent on descriptive studies and models of IGFBP overexposure in vitro and in vivo. In the literature, the role of the IGFBPs for bone growth is highly controversial and, to date, no systematic look has been taken at IGFBPs resolving functional aspects of IGFBPs at levels of cell types and specific locations within bones. Since IGFBPs are thought to represent local modulators of the IGF actions and also exert IGF-independent effects, this approach is particularly reasonable on a physiological level. By sorting the huge number of in part controversial results on IGFBP effects in bone present in the literature for distinct cell types and bone sites it is possible to generate a focused, more specific and a less controversial picture of IGFBP functions in bone