Novel and de novo PKD1 mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)

BACKGROUND: We have previously developed a long RT-PCR method for selective amplification of full-length PKD1 transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the PKD1 gene. These have provided us with an opportunity to study PKD1 mutations especially in its reiterated region which is difficult to examine. In this report, we have further developed the method of multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP) for analysis of PKD1 mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and de novo PKD1 mutations are identified and reported. METHODS: Full-length PKD1 cDNA isolated from the patients with ADPKD was fractionated into nine overlapping segments by nested-PCR. Each segment was digested with sets of combined restriction endonucleases before the SSCP analysis. The fragments with aberrant migration were mapped, isolated, and sequenced. The presence of mutation was confirmed by the long-range genomic DNA amplification in the PKD1 region, sequencing, direct mutation detection, and segregation analysis in the affected family. RESULTS: Five PKD1 mutations identified are two frameshift mutations caused by two di-nucleotide (c. 5225_5226delAG and c.9451_9452delAT) deletions, a nonsense (Q1828X, c.5693C>T) mutation, a splicing defect attributable to 31 nucleotide deletion (g.33184_33214del31), and an in-frame deletion (L3287del, c.10070_10072delCTC). All mutations occurred within the reiterated region of the gene involving exons 15, 26, 15, 19 and 29, respectively. Three mutations (one frameshift, splicing defect, and in-frame deletion) are novel and two (one frameshift and nonsense) known. In addition, two mutations (nonsense and splicing defect) are possibly de novo. CONCLUSION: The MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening PKD1 mutations in the full-length cDNA. Five mutations identified were all in the reiterated region of this gene, three of which were novel. The presence of de novo PKD1 mutations indicates that this gene is prone to mutations

Genome-Wide Analysis of Gene Expression in Primate Taste Buds Reveals Links to Diverse Processes

Efforts to unravel the mechanisms underlying taste sensation (gustation) have largely focused on rodents. Here we present the first comprehensive characterization of gene expression in primate taste buds. Our findings reveal unique new insights into the biology of taste buds. We generated a taste bud gene expression database using laser capture microdissection (LCM) procured fungiform (FG) and circumvallate (CV) taste buds from primates. We also used LCM to collect the top and bottom portions of CV taste buds. Affymetrix genome wide arrays were used to analyze gene expression in all samples. Known taste receptors are preferentially expressed in the top portion of taste buds. Genes associated with the cell cycle and stem cells are preferentially expressed in the bottom portion of taste buds, suggesting that precursor cells are located there. Several chemokines including CXCL14 and CXCL8 are among the highest expressed genes in taste buds, indicating that immune system related processes are active in taste buds. Several genes expressed specifically in endocrine glands including growth hormone releasing hormone and its receptor are also strongly expressed in taste buds, suggesting a link between metabolism and taste. Cell type-specific expression of transcription factors and signaling molecules involved in cell fate, including KIT, reveals the taste bud as an active site of cell regeneration, differentiation, and development. IKBKAP, a gene mutated in familial dysautonomia, a disease that results in loss of taste buds, is expressed in taste cells that communicate with afferent nerve fibers via synaptic transmission. This database highlights the power of LCM coupled with transcriptional profiling to dissect the molecular composition of normal tissues, represents the most comprehensive molecular analysis of primate taste buds to date, and provides a foundation for further studies in diverse aspects of taste biology

Using a modified Delphi methodology to gain consensus on the use of dressings in chronic wounds management

Objective: Managing chronic wounds is associated with a burden to patients, caregivers, health services and society and there is a lack of clarity regarding the role of dressings in improving outcomes. This study aimed to provide understanding on a range of topics, including: the definition of chronicity in wounds, the burden of illness, clinical outcomes of reducing healing time and the impact of early interventions on clinical and economic outcomes and the role of matrix metalloproteinases (MMPs) in wound healing. Method: A systematic review of the literature was carried out on the role of dressings in diabetic foot ulcer (DFU), and venous leg ulcer (VLU) management strategies, their effectiveness, associated resource use/cost, and quality of life (QoL) impact on patients. From this evidence-base statements were written regarding chronicity in wounds, burden of illness, healing time, and the role of MMPs, early interventions and dressings. A modified Delphi methodology involving two iterations of email questionnaires followed by a face-to-face meeting was used to validate the statements, in order to arrive at a consensus for each. Clinical experts were selected, representing nurses, surgeons, podiatrists, academics, and policy experts. Results: In the first round, 38/47 statements reached or exceeded the consensus threshold of 80% and none were rejected. According to the protocol, any statement not confirmed or rejected had to be modified using the comments from participants and resubmitted. In the second round, 5/9 remaining statements were confirmed and none rejected, leaving 4 to discuss at the meeting. All final statements were confirmed with at least 80% consensus. Conclusion: This modified Delphi panel sought to gain clarity from clinical experts surrounding the use of dressings in the management of chronic wounds. A full consensus statement was developed to help clinicians and policy makers improve the management of patients with these conditions