PTPRF is disrupted in a patient with syndromic amastia

<p>Abstract</p> <p>Background</p> <p>The presence of mammary glands distinguishes mammals from other organisms. Despite significant advances in defining the signaling pathways responsible for mammary gland development in mice, our understanding of human mammary gland development remains rudimentary. Here, we identified a woman with bilateral amastia, ectodermal dysplasia and unilateral renal agenesis. She was found to have a chromosomal balanced translocation, 46,XX,t(1;20)(p34.1;q13.13). In addition to characterization of her clinical and cytogenetic features, we successfully identified the interrupted gene and studied its consequences.</p> <p>Methods</p> <p>Characterization of the breakpoints was performed by molecular cytogenetic techniques. The interrupted gene was further analyzed using quantitative real-time PCR and western blotting. Mutation analysis and high-density SNP array were carried out in order to find a pathogenic mutation. Allele segregations were obtained by haplotype analysis.</p> <p>Results</p> <p>We enabled to identify its breakpoint on chromosome 1 interrupting the <it>protein tyrosine receptor type F gene </it>(<it>PTPRF</it>). While the patient's mother and sisters also harbored the translocated chromosome, their non-translocated chromosomes 1 were different from that of the patient. Although a definite pathogenic mutation on the paternal allele could not be identified, <it>PTPRF</it>'s RNA and protein of the patient were significantly less than those of her unaffected family members.</p> <p>Conclusions</p> <p>Although <it>ptprf </it>has been shown to involve in murine mammary gland development, no evidence has incorporated <it>PTPRF </it>in human organ development. We, for the first time, demonstrated the possible association of <it>PTPRF </it>with syndromic amastia, making it a prime candidate to investigate for its spatial and temporal roles in human breast development.</p

Disease-Causing 7.4 kb Cis-Regulatory Deletion Disrupting Conserved Non-Coding Sequences and Their Interaction with the FOXL2 Promotor: Implications for Mutation Screening

To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5′ to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular

Research final report production and characterization of chimeric transthyretin scavenger for amyloidosis and alzheimer's disease related proteins

Thai Governmen

Interaction between chimeric TTR and protein(s) in CSF, and its effect on abilities of chimeric TTR to inhibit and disrupt amyloid B

Alzheimer's disease (AD), one of the most frequent types of amyloidosis, is commonly associates with dementia in human. The extracellular deposition as amyloid plaque and toxicity to cells of amyloid B (AB) are common causes. Thus, removal of AB is the most important therapeutic strategy for AD. Besides function as a distributor for thyroid hormones (THS) and retinol, transthyretin (TTR) contains a proteolytic activity and it is known as a major AB sequestering protein in human brain. Recently, chimeric TTRs with higher proteolytic activity than wild type TTR were constructed in our laboratory. To ensure these TTRs can be used as potent therapeutic agents, we investigated their proteolytic activities both in vitro and in vivo. In addition, to explore a possible effect of particular proteins in the CSF such as metallothinein (MT) on the degradation of AB, the experiment was also carried with the presence of the CSF protein. The recombinant chimeric TTRs including xenoN/croc TTR and pigC/croc TTR were successfully synthesized and secreted by using the heterologous gene expression system of Pichia pastoris. The purification by preparative discontinuous native-PAGE showed only a single band with a migration to the position corresponding to TTR. The synthesized TTRs contain the physicochemical characteristics similar to those observed in the native TTRs. By using FITC-casein as substrate, the results showed that crocTTR with longer N- or C-terminal sequence had higher catalytic activity than wild type crocTTR, indicating to the effect of N- and C-terminal sequences on the activity of TTR. In addition, neither the presence of metallothionein 1 nor metallothionein 2 (MT1 and MT2) changed the AB1-42 degradation activity of human TTR. On the other hand, the degradation of AB 142 by the two chimeric TTRs was enhanced with the presence of either MTI or MT2. We also determined whether the two chimeric TTRs toxic to cells and/or have the protective effect on the toxicity of AB. The cytotoxicity assay was conducted using neuroblastoma as cell target and the cell viability was determined by MTT assay. The results showed that the viability of cells with the presence of TTR was significantly higher than the control in which cell was treated with AB alone. In comparison with human TTR, crocTTR and chimeric TTR (either xenoN/crocTTR or pigC/crocTTR) showed more protective effect. However, there was no significantly difference of the effect between xenoN/crocTTR and pigC/crocTTR. This indicated to non-toxic but ability to protect cell of the chimeric TTRS

Fabrication of protein microarrays for alpha fetoprotein detection by using a rapid photo-immobilization process

In this study, protein microarrays based on sandwich immunoassays are generated to quantify the amount of alpha fetoprotein (AFP) in blood serum. For chip generation a mixture of capture antibody and a photoactive copolymer consisting of N,N-dimethylacrylamide (DMAA), methacryloyloxy benzophenone (MaBP), and Na-4-styrenesulfonate (SSNa) was spotted onto unmodified polymethyl methacrylate (PMMA) substrates. Subsequently to printing of the microarray, the polymer and protein were photochemically cross-linked and the forming, biofunctionalized hydrogels simultaneously bound to the chip surface by short UV- irradiation. The obtained biochip was incubated with AFP antigen, followed by biotinylated AFP antibody and streptavidin-Cy5 and the fluorescence signal read-out. The developed microarray biochip covers the range of AFP in serum samples such as maternal serum in the range of 5 and 100 ng/ml. The chip production process is based on a fast and simple immobilization process, which can be applied to conventional plastic surfaces. Therefore, this protein microarray production process is a promising method to fabricate biochips for AFP screening processes. Keywords: Photo-immobilization, Protein microarray, Alpha fetoprotein, Hydrogel, 3D surface, Down syndrom

Fabrication of protein microarrays for alpha fetoprotein detection by using a rapid photo-immobilization process

In this study, protein microarrays based on sandwich immunoassays are generated to quantify the amount of alpha fetoprotein (AFP) in blood serum. For chip generation a mixture of capture antibody and a photoactive copolymer consisting of N,N-dimethylacrylamide (DMAA), methacryloyloxy benzophenone (MaBP), and Na-4-styrenesulfonate (SSNa) was spotted onto unmodified polymethyl methacrylate (PMMA) substrates. Subsequently to printing of the microarray, the polymer and protein were photochemically cross-linked and the forming, biofunctionalized hydrogels simultaneously bound to the chip surface by short UV- irradiation. The obtained biochip was incubated with AFP antigen, followed by biotinylated AFP antibody and streptavidin-Cy5 and the fluorescence signal read-out. The developed microarray biochip covers the range of AFP in serum samples such as maternal serum in the range of 5 and 100 ng/ml. The chip production process is based on a fast and simple immobilization process, which can be applied to conventional plastic surfaces. Therefore, this protein microarray production process is a promising method to fabricate biochips for AFP screening processes. Keywords: Photo-immobilization, Protein microarray, Alpha fetoprotein, Hydrogel, 3D surface, Down syndrom