Home About us Contact
|
Home About us Contact | ||
|
|
||
Pathological Mutations (pathological + mutation)
Selected AbstractsMultiplex primer extension analysis for rapid detection of major European mitochondrial haplogroupsELECTROPHORESIS, Issue 19 2006Martina Wiesbauer Abstract The evolution of the human mitochondrial genome is reflected in the existence of ethnically distinct lineages or haplogroups. Alterations of mitochondrial DNA (mtDNA) have been instrumental in studies of human phylogeny, in population genetics, and in molecular medicine to link pathological mutations to a variety of human diseases of complex etiology. For each of these applications, rapid and cost effective assays for mtDNA haplogrouping are invaluable. Here we describe a hierarchical system for mtDNA haplogrouping that combines multiplex PCR amplifications, multiplex single-base primer extensions, and CE for analyzing ten haplogroup-diagnostic mitochondrial single nucleotide polymorphisms. Using this rapid and cost-effective mtDNA genotyping method, we were able to show that within a large, randomly selected cohort of healthy Austrians (n,=,1172), mtDNAs could be assigned to all nine major European haplogroups. Forty-four percent belonged to haplogroup H, the most frequent haplogroup in European Caucasian populations. The other major haplogroups identified were U (15.4%), J (11.8%), T (8.2%) and K (5.1%). The frequencies of haplogroups in Austria is within the range observed for other European countries. Our method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and for identifying markers of genetic susceptibility. [source] The 28-amino acid form of an APLP1-derived A,-like peptide is a surrogate marker for A,42 production in the central nervous systemEMBO MOLECULAR MEDICINE, Issue 4 2009Kanta Yanagida Abstract Surrogate markers for the Alzheimer disease (AD)-associated 42-amino acid form of amyloid-, (A,42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1-derived A,-like peptides (APL1,) that are generated by ,- and ,-cleavages at a concentration of ,4.5,nM. These novel peptides, APL1,25, APL1,27 and APL1,28, were not deposited in AD brains. Interestingly, most ,-secretase modulators (GSMs) and familial AD-associated presenilin1 mutants that up-regulate the relative production of A,42 cause a parallel increase in the production of APL1,28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1,28 levels are higher than in non-AD controls, while the relative A,42 levels are unchanged or lower. Most strikingly, the relative APL1,28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non-AD controls. Based on these results, we propose the relative level of APL1,28 in the CSF as a candidate surrogate marker for the relative level of A,42 production in the brain. [source] Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling,HUMAN MUTATION, Issue 6 2009Steven F. Dobrowolski Abstract Identifying mitochondrial DNA (mtDNA) sequence variants in human diseases is complicated. Many pathological mutations are heteroplasmic, with the mutant allele represented at highly variable percentages. High-resolution melt (HRM or HRMA) profiling was applied to comprehensive assessment of the mitochondrial genome and targeted assessment of recognized pathological mutations. The assay panel providing comprehensive coverage of the mitochondrial genome utilizes 36 overlapping fragments (301,658,bp) that employ a common PCR protocol. The comprehensive assay identified heteroplasmic mutation in 33 out of 33 patient specimens tested. Allele fraction among the specimens ranged from 1 to 100%. The comprehensive assay panel was also used to assess 125 mtDNA specimens from healthy donors, which identified 431 unique sequence variants. Utilizing the comprehensive mtDNA panel, the mitochondrial genome of a patient specimen may be assessed in less than 1 day using a single 384-well plate or two 96-well plates. Specific assays were used to identify the myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) mutation m.3243A>G, myoclonus epilepsy, ragged red fibers (MERRF) mutation m.8344A>G, and m.1555A>G associated with aminoglycoside hearing loss. These assays employ a calibrated, amplicon-based strategy that is exceedingly simple in design, utilization, and interpretation, yet provides sensitivity to detect variants at and below 10% heteroplasmy. Turnaround time for the genotyping tests is about 1,hr. Hum Mutat 30,1,8, 2009. © 2009 Wiley-Liss, Inc. [source] Oculocutaneous albinism type 4: six novel mutations in the membrane-associated transporter protein gene and their phenotypesPIGMENT CELL & MELANOMA RESEARCH, Issue 5 2006Katsuhiko Inagaki Summary Oculocutaneous albinism type 4 (OCA4) is an autosomal recessive hypopigmentary disorder caused by mutations in the Membrane-Associated Transporter Protein gene (SLC45A2). The SLC45A2 protein is a 530-amino-acid polypeptide that contains 12 putative transmembrane domains, and appears to be a transporter that mediates melanin synthesis. Eighteen pathological mutations have been reported so far. In this study, six novel mutations, p.Y49C (c.146A > G), p.G89R (c.265G > A), p.C229Y (c.686G > A), p.T437A (c.1309A > G), p.T440A (c.1318A > G) and p.G473D (c.1418G > A) were found in eight Japanese patients with various clinical phenotypes. The phenotypes of OCA4 were as various as the other types of OCA and probably depended on the mutation sites in the SLC45A2 gene. [source] The strong dimerization of the transmembrane domain of the fibroblast growth factor receptor (FGFR) is modulated by C-terminal juxtamembrane residuesPROTEIN SCIENCE, Issue 2 2009Weng Chuan Peng Abstract The fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR subfamily of the receptor tyrosine kinases (RTKs) involved in signaling across the plasma membrane. Generally, ligand binding leads to receptor dimerization and activation. Dimerization involves the transmembrane (TM) domain, where mutations can lead to constitutive activation in certain cancer types and also in skeletal malformations. Thus, it has been postulated that FGFR homodimerization must be inherently weak to allow regulation, a feature reminiscent of , and , integrin TM interactions. However, we show herein that in FGFR3-TM, four C-terminal residues, CRLR, have a profound destabilizing effect in an otherwise strongly dimerizing TM peptide. In the absence of these four residues, the dimerizing propensity of FGFR3-TM is comparable to glycophorin, as shown using various detergents. In addition, the expected enhanced dimerization induced by the mutation associated to the Crouzon syndrome A391E, was observed only when these four C-terminal residues were present. In the absence of these four residues, A391E was dimer-destabilizing. Finally, using site specific infrared dichroism and convergence with evolutionary conservation data, we have determined the backbone model of the FGFR3-TM homodimer in model lipid bilayers. This model is consistent with, and correlates with the effects of, most known pathological mutations found in FGFR-TM. [source] The production ratios of AICD,51 and A,42 by intramembrane proteolysis of ,APP do not always change in parallelPSYCHOGERIATRICS, Issue 3 2010Kohji MORI Abstract Background:, During intramembrane proteolysis of ,-amyloid protein precursor (,APP) by presenilin (PS)/,-secretase, ,-cleavages at the membrane-cytoplasmic border precede ,-cleavages at the middle of the transmembrane domain. Generation ratios of A,42, a critical molecule for Alzheimer's disease (AD) pathogenesis, and the major A,40 species might be associated with ,48 and ,49 cleavages, respectively. Medicines to downregulate A,42 production have been investigated by many pharmaceutical companies. Therefore, the ,-cleavages, rather than the ,-cleavage, might be more effective upstream targets for decreasing the relative generation of A,42. Thus, one might evaluate compounds by analyzing the generation ratio of the ,APP intracellular domain (AICD) species (,-cleavage-derived), instead of that of A,42. Methods:, Cell-free ,-secretase assays were carried out to observe de novo AICD production. Immunoprecipitation/MALDI-TOF MS analysis was carried out to detect the N-termini of AICD species. A, and AICD species were measured by ELISA and immunoblotting techniques. Results:, Effects on the ,-cleavage by AD-associated pathological mutations around the ,-cleavage sites (i.e., ,APP V642I, L648P and K649N) were analyzed. The V642I and L648P mutations caused an increase in the relative ratio of ,48 cleavage, as expected from previous reports. Cells expressing the K649N mutant, however, underwent a major ,-cleavage at the ,51 site. These results suggest that ,51, as well as ,48 cleavage, is associated with A,42 production. Only AICD,51, though, and not A,42 production, dramatically changed with modifications to the cell-free assay conditions. Interestingly, the increase in the relative ratio of the ,51 cleavage by the K649N mutation was not cancelled by these changes. Conclusion:, Our current data show that the generation ratio of AICD,51 and A,42 do not always change in parallel. Thus, to identify compounds that decrease the relative ratio of A,42 generation, measurement of the relative level of A,42-related AICD species (i.e., AICD,48 and AICD,51) might not be useful. Further studies to reveal how the ,-cleavage precision is decided are necessary before it will be possible to develop drugs targeting ,-cleavage as a means for decreasing A,42 production. [source] | ||||||||||