Wyniki wyszukiwania

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Sekwencjonowanie DNA nowej generacji (NGS) – zastosowanie w diagnostyce

100%

Ploski R.

Diagnostyka Laboratoryjna

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2017

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tom 53

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nr 1A

2

Human induced pluripotent stem cell (HIPSCS) based Huntington’s disease (HD) neurons and their genetic correction using personalized RNA-guided designer nucleases (CAS9) mediated genome editing

60%

Golonko A., Pollak A., Rydzanicz M., Ploski R., Lisowski P.

Acta Neurobiologiae Experimentalis

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2017

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tom 77

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nr Suppl.1

INTRODUCTION: Huntington’s Disease (HD) is an neurodegenerative disorder, caused by a mutation in the CAG repeat tract of the huntingtin gene HTT, characterized by a progressive loss of neurons in the striatum and phenotypic features that are common in other neurodegenerative diseases. AIM(S): The aim was to 1) test personalized, precise genome editing technology based on high fidelity Cas9 variants in somatic gene therapy of trinucleotide repeat degenerative disorder such as HD, 2) and to test whether the N-terminal truncated protein is able to support normal neuronal development, 3) dissect the impact of the mutation on neuronal development. METHOD(S): Patient specific hiPSCs were generated using an integration-free method. hiPSC were edited by improved fidelity Cas9‑sgRNA expression vectors located upstream and downstream of the CAG repeats in Exon 1, HDR repair templates with different numbers of CAG repeats. Gene edited iPS clones were characterized for the potential modification at predicted off‑target sites. The lines were subjected for the functional studies with high-content screening. RESULTS: hiPSCs editing resulted into different products that underwent the non-homologous end joining (NHEJ), precisely corrected clones by homologous recombination (HR) and NHEJ mediated excision of the Q/P repeat region by reannealing of the DSB resulted into an in-frame Htt coding region lacking the N‑terminal Q/P repeat. CONCLUSIONS: 1) HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active neurons; 2) Study demonstrates associated phenotypic abnormalities; 3) Personalized high fidelity Cas9 variants showed an improved specificity profile, suitable for somatic gene therapy; 4) Study shows the importance of isogenic controls for modeling and personalized gene therapy using patient specific hiPSCs. FINANCIAL SUPPORT: Acknowledgements: National Science Centre (Poland) Grant No. 2016/22/M/NZ2/00548 to PL.

3

Mild Zellweger syndrome due to a novel PEX6 mutation: correlation between clinical phenotype and in silico prediction of variant pathogenicity

51%

Rydzanicz M., Stradomska T. J., Jurkiewicz E., Jamroz E., Gasperowicz P., Kostrzewa G., Ploski R.

Journal of Applied Genetics

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2017

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tom 58

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nr 4

4

ARMS-PCR for detection of BRAF V600E hotspot mutation in comparison with Real-Time PCR-based techniques

51%

Machnicki M. M., Glodkowska-Mrowka E., Lewandowski T., Ploski R., Wlodarski P., Stoklosa T.

Acta Biochimica Polonica

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2013

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tom 60

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nr 1

BRAF mutation testing is one of the best examples how modern genetic testing may help to effectively use targeted therapies in cancer patients. Since many different genetic techniques are employed to assess BRAF mutation status with no; available comparison of their sensitivity and usefulness for different types of samples, we decided to evaluate our own PCR-based assay employing the amplification refractory mutation system (ARMS-PCR) to detect the most common hotspot mutation c. T1799A (p. V600E) by comparing it with two qPCR based assays: a commercially available test with hybridizing probes (TIB MOLBIOL) and high resolution melting (HRM). Positive results were verified with Sanger sequencing. DNA from two cancer cell lines with known mutation status and from tissue samples from melanoma and gastric cancer was used. ARMS-PCR was the most sensitive method with the level of detection of the mutant allele at 2%. Similar sensitivity was observed for the qPCR-based commercial test employing hybridizing probes; however, this test cannot exclude negative results from poor or low quality samples. Another qPCR-based method, HRM, had lower sensitivity with the detection level of approximately 20%. An additional drawback of HRM methodology was the inability to distinguish between wild type and mutant homozygotes in a straightforward assay, probably due to the character of this particular mutation (T>A). Sanger sequencing had the sensitivity of the detection of mutant allele similar to HRM, approx. 20%. In conclusion, simple ARMS-PCR may be considered the method of choice for rapid, cost-effective screening for BRAF p. V600E mutation.

5

Genetics of mitochondrial diseases – from mitochondrial DNA to whole exome studies

43%

Tonska K., Kaliszewska M., Rusecka J., Kierdaszuk B., Rydzanicz M., Stawinski P., Ploski R., Kaminska A.

Acta Neurobiologiae Experimentalis

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2017

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tom 77

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nr Suppl.1

Mitochondrial diseases, caused by dysfunction of the respiratory chain are characterised by very high clinical as well as genetic heterogeneity. In most of the cases multiple organs and systems are involved with special place taken by muscular and nervous systems due to their high respiratory requirements. From the genetic point of view mitochondrial diseases are exceptionally difficult to study. As the respiratory chain function is secured by the cooperation of up to 1500 proteins, the number of genes in which mutations may lead to OXPHOS dysfunction may be close to that number. Another difficulty is that the respiratory chain subunits are encoded by two different genomes. 13 of them are localised in mitochondrial DNA (mtDNA) – small, multicopy maternally inherited molecule. The remaining 70 are nuclear encoded. This means that the mutations responsible for mitochondrial diseases may be inherited both in a mendelian and a maternal way. A group of the diseases caused by mutations in nuclear genes encoding proteins responsible for mtDNA maintenance is worth mentioning. mtDNA depletion or multiple deletions are observed as a result of such mutations. POLG and C10orf2 mutations are the most frequent in Polish patients. Next generation sequencing (NGS) enabled detailed analysis of both genomes. The application of NGS to mtDNA analysis in our hands has proven to be an effective tool to capture known as well as novel pathogenic variants. Due to the high number of reads and high coverage it also allows the detection of low levels of heteroplasmy. WES was applied to analyse genetic background of the disease in adult patients with progressive external ophthalmoplegia, multiple mtDNA deletions and negative screening for POLG and C10orf2 mutations. The preliminary results indicate that the success ratio is much lower than in paediatric patients. FINANCIAL SUPPORT: This work was supported by the National Science Center of Poland grant 2014/15/B/ NZ2/02272.

6

Nuclear genes involved in mitochondrial diseases with mitochondrial DNA instability in adults

35%

Rusecka J., Kierdaszuk B., Kaliszewska M., Rydzanicz M., Stawinski P., Gambin T., Kostera-Pruszczyk A., Bartnik E., Kaminska A., Ploski R., Tonska K.

Acta Biochimica Polonica

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2018

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tom 65

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nr S2

7

Neonatal cholestasis due to citrin deficiency: diagnostic pitfalls

35%

Lipinski P., Jurkiewicz D., Ciara E., Ploski R., Wiecek S., Bogdanska A., Stradomska T., Socha P., Rokicki D., Tylki-Szymanska A., Jankowska I.

Acta Biochimica Polonica

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2020

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tom 67

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nr 2

8

Novel sporadic and recurrent mutations in KRT5 and KRT14 genes in Polish epidermolysis bullosa simplex patients: further insights into epidemiology and genotype–phenotype correlation

35%

Wertheim-Tysarowska K., Oldak M., Giza A., Kutkowska-Kazmierczak A., Sota J., Przybylska D., Wozniak K., Sniegorska D., Niepokoj K., Sobczynska-Tomaszewska A., Rygiel A. M., Ploski R., Bal J., Kowalewski C.

Journal of Applied Genetics

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2016

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tom 57

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nr 2

9

Genetic and environmental predictors of chronic kidney disease in patients with type 2 diabetes and diabetic foot ulcer: a pilot study

35%

Mrozikiewicz-Rakowska B., Maroszek P., Nehring P., Sobczyk-Kopciol A., Krzyzewska M., Kaszuba A. M., Lukawska M., Chojnowska N., Kozka M., Bujalska-Zadrozny M., Ploski R., Krzymien J., Czupryniak L.

Journal of Physiology and Pharmacology

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2015

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tom 66

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nr 5

Ograniczanie wyników

Sekwencjonowanie DNA nowej generacji (NGS) – zastosowanie w diagnostyce

Human induced pluripotent stem cell (HIPSCS) based Huntington’s disease (HD) neurons and their genetic correction using personalized RNA-guided designer nucleases (CAS9) mediated genome editing

Mild Zellweger syndrome due to a novel PEX6 mutation: correlation between clinical phenotype and in silico prediction of variant pathogenicity

ARMS-PCR for detection of BRAF V600E hotspot mutation in comparison with Real-Time PCR-based techniques

Genetics of mitochondrial diseases – from mitochondrial DNA to whole exome studies

Nuclear genes involved in mitochondrial diseases with mitochondrial DNA instability in adults

Neonatal cholestasis due to citrin deficiency: diagnostic pitfalls

Novel sporadic and recurrent mutations in KRT5 and KRT14 genes in Polish epidermolysis bullosa simplex patients: further insights into epidemiology and genotype–phenotype correlation

Genetic and environmental predictors of chronic kidney disease in patients with type 2 diabetes and diabetic foot ulcer: a pilot study