NQO1 expression correlates inversely with NFκB activation in human breast cancer

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A Transgenic Minipig Model of Huntington\u27s Disease

Background: Some promising treatments for Huntington\u27s disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. Objective: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1–548 under the control of human HTT promoter. Methods: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. Results: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. Conclusions: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study

Analyza mutaci u pacientu s tuberozni sklerozou.

Tuberous sclerosis complex (TSC) is an autosomal neurocutaneous disorder with a very variable clinical picture. It is characterized by development of benign tumours (hamartomas) affecting many organ systems including skin, central nervous system, kidney and heart. The most important clinical signs are epilepsy, mental retardation, facial angiofibromas, hypomelanotic patches and renal angiomyolipomas.The estimated prevalence of the disease is about 1:10000 live births. About 2/3 of affected patients are thought to have new mutations. The disease is caused by mutation of two involved genes, either on chromosome 9q34 (TSC1 gene) or on chromosome 16p13.3 (TSC2 gene). DNA mutation analysis is effective namely in families with multiple TSC incidence. In such families the linkage can be used and there are more TSC1 cases which are easily identifiable. Analysis is not economical for differential diagnosis. The number of revealed mutations correspondes to equal distribution of mutations in genes, with higher incidence in TSC2 gene. No correlation between the phenotype and the type of mutation was found. Genetic consultation have to be aware of the risk of mozaicism.Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

Detection of Unknown and Rare Pathogenic Variants in Antithrombin, Protein C and Protein S Deficiency Using High-Throughput Targeted Sequencing

The deficiency of natural anticoagulants—antithrombin (AT), protein C (PC), and protein S (PS)—is a highly predisposing factor for thrombosis, which is still underdiagnosed at the genetic level. We aimed to establish and evaluate an optimal diagnostic approach based on a high-throughput sequencing platform suitable for testing a small number of genes. A fast, flexible, and efficient method involving automated amplicon library preparation and target sequencing on the Ion Torrent platform was optimized. The cohort consisted of a group of 31 unrelated patients selected for sequencing due to repeatedly low levels of one of the anticoagulant proteins (11 AT-deficient, 13 PC-deficient, and 7 PS-deficient patients). The overall mutation detection rate was 67.7%, highest in PC deficiency (76.9%), and six variants were newly detected—SERPINC1 c.398A > T (p.Gln133Leu), PROC c.450C > A (p.Tyr150Ter), c.715G > C (p.Gly239Arg) and c.866C > G (p.Pro289Arg), and PROS1 c.1468delA (p.Ile490fs) and c.1931T > A (p.Ile644Asn). Our data are consistent with those of previous studies, which mostly used time-consuming Sanger sequencing for genotyping, and the indication criteria for molecular genetic testing were adapted to this process in the past. Our promising results allow for a wider application of the described methodology in clinical practice, which will enable a suitable expansion of the group of indicated patients to include individuals with severe clinical findings of thrombosis at a young age. Moreover, this approach is flexible and applicable to other oligogenic panels

High-Throughput Sequencing Haplotype Analysis Indicates in LRRK2 Gene a Potential Risk Factor for Endemic Parkinsonism in Southeastern Moravia, Czech Republic

Parkinson’s disease and parkinsonism are relatively common neurodegenerative disorders. This study aimed to assess potential genetic risk factors of haplotypes in genes associated with parkinsonism in a population in which endemic parkinsonism and atypical parkinsonism have recently been found. The genes ADH1C, EIF4G1, FBXO7, GBA, GIGYF2, HTRA2, LRRK2, MAPT, PARK2, PARK7, PINK1 PLA2G6, SNCA, UCHL1, and VPS35 were analyzed in 62 patients (P) and 69 age-matched controls from the researched area (C1). Variants were acquired by high-throughput sequencing using Ion Torrent workflow. As another set of controls, the whole genome sequencing data from 100 healthy non-related individuals from the Czech population were used (C2); the results were also compared with the Genome Project data (C3). We observed shared findings of four intron (rs11564187, rs36220738, rs200829235, and rs3789329) and one exon variant (rs33995883) in the LRRK2 gene in six patients. A comparison of the C1–C3 groups revealed significant differences in haplotype frequencies between ratio of 2.09 for C1, 1.65 for C2, and 6.3 for C3, and odds ratios of 13.15 for C1, 2.58 for C2, and 7.6 for C3 were estimated. The co-occurrence of five variants in the LRRK2 gene (very probably in haplotype) could be an important potential risk factor for the development of parkinsonism, even outside the recently described pedigrees in the researched area where endemic parkinsonism is present

Giant angiofibromas in tuberous sclerosis complex: A possible role for localized lymphedema in their pathogenesis

BACKGROUND: Giant angiofibromas in patients with tuberous sclerosis complex (TSC) are rare. OBJECTIVE: We sought to report two patients who had TSC with unusually large and disfiguring facial angiofibromas and to identify underlying histopathologic changes that may possibly explain the clinical features. METHODS: We performed a clinicopathologic, immunohistochemical, and molecular biologic study using 42 lesional specimens and peripheral blood from one of the two patients. The immunohistochemical investigations were mainly focused on the vascular moiety of the lesions. TSC1 and TSC2 alterations were studied using multiplex ligation-dependent probe amplification for large deletion/duplication mutations, whereas screening for small mutations was performed using polymerase chain reaction amplification of individual coding exons and exon-intron junctions of both genes followed by an analysis on denaturation gradient gel electrophoresis. RESULTS: Histopathologic examination revealed, in addition to findings typical of angiofibroma, several unusual features including multinucleated giant cells containing multiple intracytoplasmic vacuoles, Touton-like cells, emperipolesis, pagetoid dyskeratosis, vacuolar alteration at the dermoepidermal junction, Civatte bodies, and melanophages in the subjacent dermis. Numerous dilated lymphatic vessels were detected indicating localized lymphostasis, probably caused by secondary lymphedema. The lymphatic nature of the vessels was confirmed by immunohistochemical study. Genetic testing for TSC1 and TSC2 gene mutations revealed a substitution on position c.2251C>T resulting in a nonsense mutation R751X in fragment 20.2. LIMITATIONS: Histopathologic specimens and peripheral blood were available from only one patient. CONCLUSION: Localized lymphedema may contribute to the formation of large disfiguring angiofibromas in patients with TSC