Abstract: We have studied the association of three single nucleotide polymorphisms (SNPs) present in the three HSP70 (heat‐shock protein) genes on 6p21 with human longevity. The availability of biological samples from various population cohorts in Denmark has given us the opportunity to try novel methods of gene association with human longevity. A significant association of one haplotype with male longevity was observed. Furthermore, a significant difference in the survival of the carriers of the different genotypes in females was observed. We also found an age‐dependant decline in the ability of peripheral blood mononuclear cells to respond to heat stress in terms of Hsp70 induction.
Reverse transcription-PCR (RT-PCR), combined with an endogenous and exogenous standard, is used extensively to quantify specific mRNAs, but few studies have evaluated the reproducibility of these assays (1)(2)(3)(4)(5)(6)(7). Increased expression of the epidermal growth factor receptor (EGFr) has been observed in several human cancers and has been suggested to predict tumor progression (8)(9)(10)(11). Quantitative analysis for the EGFr mRNA using competitive RT-PCR has been described (12)(13)(14), but the analytical imprecision has not yet been documented systematically.
A primer pair was designed to flank the first intron of the human EGFr gene (15)(16). The 350-bp cDNA fragment was amplified as described below, using the following primers: primer I (5′-GAC CCT CCG GGA CGG-3′), spanning positions 191–205; and primer II (5′-GGC ATA GGA ATT TTC GTA GTA CAT AT-3′), spanning positions 515–540 and cloned into a pCRTM II vector (TA Cloning System, Invitrogen). The “EGFr” plasmid was purified using a Qiagen plasmid midi kit (Qiagen, Inc.) and cleaved by Bgl II (Pharmacia Biotech). A 69-bp deletion was obtained by PCR (see below) using the linear plasmid as template, primer III (5′-GAC CCT CCG GGA CGG CCG GGC AAG GCA CGA GTA ACA AGC TCA CGC AGT TGG 3′), and primer II (Fig. 1⇓ A). The PCR product was cloned into a pCR II vector, purified, and cleaved as above. The PCR products of the EGFr and standard were sequenced on both strands, confirming their identity and the absence of PCR errors (data not shown).
To synthesize the RNA standard, 5 μg of the linear plasmid (containing the 69-bp deletion) was incubated with 100 units of SP6 RNA polymerase according to the manufacturer’s protocol (Promega Corp.). Fifty …
Metadata and statistics of integrative ChIP-seq and expression analysis. This file contains three Excel spreadsheets. Spreadsheet 1 contains information on the number of genes common to both the exon expression array and ChIPseq analyses. Spreadsheets 2 and 3 include statistical and permutation tests concerning enrichment of DEGs among BRD1 PTGs. (XLSX 12 kb)
Multiple acyl-CoA dehydrogenation deficiency is a disorder of fatty acid and amino acid oxidation caused by defects of electron transfer flavoprotein (ETF) or its dehydrogenase (ETFDH). A clear relationship between genotype and phenotype makes genotyping of patients important not only diagnostically but also for prognosis and for assessment of treatment. In the present study, we show that a predicted benign ETFDH missense variation (c.158A>G/p.Lys53Arg) in exon 2 causes exon skipping and degradation of ETFDH protein in patient samples. Using splicing reporter minigenes and RNA pull-down of nuclear proteins, we show that the c.158A>G variation increases the strength of a preexisting exonic splicing silencer (ESS) motif UAGGGA. This ESS motif binds splice inhibitory hnRNP A1, hnRNP A2/B1, and hnRNP H proteins. Binding of these inhibitory proteins prevents binding of the positive splicing regulatory SRSF1 and SRSF5 proteins to nearby and overlapping exonic splicing enhancer elements and this causes exon skipping. We further suggest that binding of hnRNP proteins to UAGGGA is increased by triggering synergistic hnRNP H binding to GGG triplets located upstream and downsteam of the UAGGGA motif. A number of disease-causing exonic elements that induce exon skipping in other genes have a similar architecture as the one in ETFDH exon 2.
Objective. To understand the expanding clinical and biochemical spectrum of short-chain acyl-CoA dehydrogenase (SCAD) deficiency, the impact of which is not fully understood. Study Design. We studied a family with SCAD deficiency and determined urinary ethylmalonic acid excretion, plasma C4-carnitine, SCAD enzyme activity in fibroblasts and lymphocytes, DNA mutations in the SCAD gene, and clinical expression. The index patient was born prematurely and had otherwise unexplained cholestasis and hepatomegaly during the first year of life. His mother developed a hemolysis-elevated liver enzymes-low platelets (HELLP) syndrome while pregnant with the index patient. Results. Two siblings had a homozygous inactivating 1138C>T mutation, whereas the father was compound heterozygous for this mutation and the common 625G>A polymorphism. There was a good correlation between the type of SCAD mutation, the residual SCAD enzyme activity, and the levels of urinary ethylmalonic acid and plasma C4-carnitine in each of the eight family members. Retrospective acylcarnitine analysis of the index patient’s Guthrie screening card confirmed the abnormal increase of C4-carnitine, suggestive of SCAD deficiency. None of the family members had hypotonia, developmental delay, or episodes of ketotic hypoglycemia. Conclusion. Homozygosity for an inactivating SCAD mutation does not necessarily result in disease. The previously held opinion that SCAD deficiency is always a serious disorder may have been influenced by a clinical bias. Homozygosity for an inactivating 1138C>T SCAD mutation was assessed by neonatal screening of blood spot acylcarnitines. SCAD deficiency may be associated with maternal HELLP syndrome.
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