Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith–Lemli–Opitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol biosynthesis pathway.
Smith-Lemli-Opitz syndrome is a recessive disorder caused by mutations in 7-dehydrocholesterol reductase (DHCR)7 with a heterozygous (HET) carrier frequency of 1-3%. A defective DHCR7 causes accumulation of 7-dehydrocholesterol (DHC), which is a highly oxidizable and toxic compound. Recent studies suggest that several antipsychotics, including the highly prescribed pharmaceuticals, aripiprazole (ARI) and trazodone (TRZ), increase 7-DHC levels in vitro and in humans. Our investigation was designed to compare the effects of ARI and TRZ on cholesterol (Chol) synthesis in fibroblasts from human carriers and controls (CTRs). Six matched pairs of fibroblasts were treated and their sterol profile analyzed by LC-MS. Significantly, upon treatment with ARI and TRZ, the total accumulation of 7-DHC was higher in-HET cells than in CTR fibroblasts. The same set of experiments was repeated in the presence of C-lanosterol to determine residual Chol synthesis, revealing that ARI and TRZ strongly inhibit de novo Chol biosynthesis. The results suggest that carriers have increased vulnerability to both ARI and TRZ exposure compared with CTRs. Thus, the 1-3% of the population who are DHCR7 carriers may be more likely to sustain deleterious health consequences on exposure to compounds like ARI and TRZ that increase levels of 7-DHC, especially during brain development.
Smith-Lemli-Opitz (SLO) syndrome is an autosomal recessive disorder characterized by multiple congenital abnormalities and mental retardation. The condition is caused by the deficiency of 7-dehydrocholesterol reductase (DHCR7) which catalyzes the final step in cholesterol biosynthesis. Biochemical diagnosis is based on increased concentration of 7-dehydrocholesterol (7-DHC) in the patient serum. Both life expectancy and quality of life are severely affected by the disease. The estimated prevalence of SLO syndrome ranges between 1:20,000 and 1:40,000 among Caucasians. Although the mutational spectrum of the disease is wide, approximately 10 mutations are responsible for more than 80% of the cases. These mutations show a large interethnic variability. There are no mutation distribution data from Hungary to date. Thirteen patients were diagnosed with SLO syndrome in our laboratory. As first-line tests, serum 7-DHC and total cholesterol were measured and, in positive cases, molecular genetic analysis of the DHCR7 gene was performed. Complete genetic background of the disease could be identified in 12 cases. In 1 case only 1 mutation was detected in a heterozygote form. One patient was homozygous for the common splice site mutation c.964–1G>C, while all other patients were compound heterozygotes. One novel missense mutation, c.374A>G (p.Tyr125Cys) was identified.
Background: In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of the FLT3 (Fms-like tyrosine kinase 3) gene is one of the most frequent genetic alterations associated with poor prognosis. Methods: A complex evaluation of the analytical properties of the three most frequently used detection methods -PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE) -was performed on 95 DNA samples obtained from 73 AML patients. Results: All the three methods verifi ed the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1 % -2 % mutant allele, while the detection limit of CE was 0.28 % . However, acceptable reproducibility (inter-assay CV < 25 % ) of the mutant allele rate determination was only achievable above 1.5 % mutant/ total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7 % lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes.
BackgroundCurrent technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis.ResultsIn the plasmid system average correct genotyping was 95.8% in 4-mers, 87.4% in 5-mers and 72.1% in 6-mers. Despite the experienced low genotyping accuracy in 5- and 6-mers, it was possible to generate amplicons with more than a 90% adequate detection rate in every homopolymer tract. When homopolymers in the CFTR gene were sequenced average accuracy was 89.3%, but varied in a wide range (52.2 – 99.1%). In all but one case, an optimal amplicon-sequencing primer combination could be identified. In that single case (7A tract in exon 14 (c.2046_2052)), none of the tested primer sets produced the required analytical performance.ConclusionsOur results show that pyrosequencing is the most reliable in case of 4-mers and as homopolymer length gradually increases, accuracy deteriorates. With careful primer selection, the NGS system was able to correctly genotype all but one of the homopolymers in the CFTR gene. In conclusion, we configured a plasmid test system that can be used to assess genotyping accuracy of NGS devices and developed an accurate NGS assay for the molecular diagnosis of CF using self-designed primers for amplification and sequencing.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4544-x) contains supplementary material, which is available to authorized users.
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