Cellular Models for Primary CoQ Deficiency Pathogenesis Study

Primary coenzyme Q10 (CoQ) deficiency includes a heterogeneous group of mitochondrial diseases characterized by low mitochondrial levels of CoQ due to decreased endogenous biosynthesis rate. These diseases respond to CoQ treatment mainly at the early stages of the disease. The advances in the next generation sequencing (NGS) as whole-exome sequencing (WES) and whole-genome sequencing (WGS) have increased the discoveries of mutations in either gene already described to participate in CoQ biosynthesis or new genes also involved in this pathway. However, these technologies usually provide many mutations in genes whose pathogenic effect must be validated. To functionally validate the impact of gene variations in the disease’s onset and progression, different cell models are commonly used. We review here the use of yeast strains for functional complementation of human genes, dermal skin fibroblasts from patients as an excellent tool to demonstrate the biochemical and genetic mechanisms of these diseases and the development of human-induced pluripotent stem cells (hiPSCs) and iPSC-derived organoids for the study of the pathogenesis and treatment approaches.

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Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

PLoS ONE ◽

10.1371/journal.pone.0260852 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0260852

Author(s):

Meryem Ozgencil ◽

Julian Barwell ◽

Marc Tischkowitz ◽

Louise Izatt ◽

Ian Kesterton ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Repair ◽

Ips Cells ◽

Variants Of Uncertain Significance ◽

Cellular Models ◽

Pathogenic Variants ◽

Damage Repair ◽

Uncertain Significance ◽

Induced Pluripotent ◽

The Impact

Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

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Whole-exome sequencing and its impact in hereditary hearing loss

Genetics Research ◽

10.1017/s001667231500004x ◽

2015 ◽

Vol 97 ◽

Cited By ~ 28

Author(s):

TAHIR ATIK ◽

GUNEY BADEMCI ◽

OSCAR DIAZ-HORTA ◽

SUSAN H. BLANTON ◽

MUSTAFA TEKIN

Keyword(s):

Hearing Loss ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Hereditary Deafness ◽

Whole Exome ◽

Human Genes ◽

Genetic Revolution ◽

Recent Developments ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

SummaryNext-generation sequencing (NGS) technologies have played a central role in the genetic revolution. These technologies, especially whole-exome sequencing, have become the primary tool of geneticists to identify the causative DNA variants in Mendelian disorders, including hereditary deafness. Current research estimates that 1% of all human genes have a function in hearing. To date, mutations in over 80 genes have been reported to cause nonsyndromic hearing loss (NSHL). Strikingly, more than a quarter of all known genes related to NSHL were discovered in the past 5 years via NGS technologies. In this article, we review recent developments in the usage of NGS for hereditary deafness, with an emphasis on whole-exome sequencing.

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Neurons derived from individual early Alzheimer′s disease patients reflect clinical vulnerability

10.1101/2021.11.09.467891 ◽

2021 ◽

Author(s):

Bryan Ng ◽

Helen Rowland ◽

Tina Wei ◽

Kanisa Arunasalam ◽

Emma Mee Hayes ◽

...

Keyword(s):

Amyloid Beta ◽

Brain Activity ◽

Activity Levels ◽

Cellular Models ◽

Novel Approach ◽

Induced Pluripotent ◽

Cellular Phenotypes ◽

The Impact

Modelling sporadic Alzheimer′s disease (sAD) with patient-derived induced pluripotent stem cells (iPSCs) is challenging yet remains an important step in understanding the disease. Here we report a novel approach of sAD modelling with patient iPSC-derived neurons by integrating cellular and clinical phenotypes from individual early symptomatic sAD patients. We establish a correlation between cellular vulnerability to extrinsic amyloid-beta in vitro measured by synapse loss with clinical vulnerability to amyloid-beta burden in vivo measured by cognitive decline and brain activity levels. Our findings indicate that patient iPSC-derived neurons not only preserve some pathological phenotypes of disease measured in the people they were derived from, but also preserve, from people to cells, the impact of those pathological phenotypes on function. Cellular models that reflect an individual′s in-life clinical vulnerability thus represent a tractable method of sAD modelling using clinical data in combination with cellular phenotypes.

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Mitochondrial Medicine: Genetic Underpinnings and Disease Modeling Using Induced Pluripotent Stem Cell Technology

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2020.604581 ◽

2021 ◽

Vol 7 ◽

Author(s):

Parisa K. Kargaran ◽

Diogo Mosqueira ◽

Tamas Kozicz

Keyword(s):

Mitochondrial Genome ◽

Disease Modeling ◽

Nuclear Genome ◽

Induced Pluripotent Stem Cell ◽

Mitochondrial Diseases ◽

Potential Candidate ◽

Cellular Models ◽

Mitochondrial Medicine ◽

Induced Pluripotent

Mitochondrial medicine is an exciting and rapidly evolving field. While the mitochondrial genome is small and differs from the nuclear genome in that it is circular and free of histones, it has been implicated in neurodegenerative diseases, type 2 diabetes, aging and cardiovascular disorders. Currently, there is a lack of efficient treatments for mitochondrial diseases. This has promoted the need for developing an appropriate platform to investigate and target the mitochondrial genome. However, developing these therapeutics requires a model system that enables rapid and effective studying of potential candidate therapeutics. In the past decade, induced pluripotent stem cells (iPSCs) have become a promising technology for applications in basic science and clinical trials, and have the potential to be transformative for mitochondrial drug development. Engineered iPSC-derived cardiomyocytes (iPSC-CM) offer a unique tool to model mitochondrial disorders. Additionally, these cellular models enable the discovery and testing of novel therapeutics and their impact on pathogenic mtDNA variants and dysfunctional mitochondria. Herein, we review recent advances in iPSC-CM models focused on mitochondrial dysfunction often causing cardiovascular diseases. The importance of mitochondrial disease systems biology coupled with genetically encoded NAD+/NADH sensors is addressed toward developing an in vitro translational approach to establish effective therapies.

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Uncovering the Diversification of Tissue Engineering on the Emergent Areas of Stem Cells, Nanotechnology and Biomaterials

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200103124821 ◽

2020 ◽

Vol 15 (3) ◽

pp. 187-201 ◽

Cited By ~ 1

Author(s):

Sunil K. Dubey ◽

Amit Alexander ◽

Munnangi Sivaram ◽

Mukta Agrawal ◽

Gautam Singhvi ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Immune System ◽

Clinical Application ◽

Cell Types ◽

Patient Specific ◽

Potential Strategy ◽

Induced Pluripotent ◽

Treat All ◽

The Impact

Damaged or disabled tissue is life-threatening due to the lack of proper treatment. Many conventional transplantation methods like autograft, iso-graft and allograft are in existence for ages, but they are not sufficient to treat all types of tissue or organ damages. Stem cells, with their unique capabilities like self-renewal and differentiate into various cell types, can be a potential strategy for tissue regeneration. However, the challenges like reproducibility, uncontrolled propagation and differentiation, isolation of specific kinds of cell and tumorigenic nature made these stem cells away from clinical application. Today, various types of stem cells like embryonic, fetal or gestational tissue, mesenchymal and induced-pluripotent stem cells are under investigation for their clinical application. Tissue engineering helps in configuring the stem cells to develop into a desired viable tissue, to use them clinically as a substitute for the conventional method. The use of stem cell-derived Extracellular Vesicles (EVs) is being studied to replace the stem cells, which decreases the immunological complications associated with the direct administration of stem cells. Tissue engineering also investigates various biomaterials to use clinically, either to replace the bones or as a scaffold to support the growth of stemcells/ tissue. Depending upon the need, there are various biomaterials like bio-ceramics, natural and synthetic biodegradable polymers to support replacement or regeneration of tissue. Like the other fields of science, tissue engineering is also incorporating the nanotechnology to develop nano-scaffolds to provide and support the growth of stem cells with an environment mimicking the Extracellular matrix (ECM) of the desired tissue. Tissue engineering is also used in the modulation of the immune system by using patient-specific Mesenchymal Stem Cells (MSCs) and by modifying the physical features of scaffolds that may provoke the immune system. This review describes the use of various stem cells, biomaterials and the impact of nanotechnology in regenerative medicine.

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Phenocopy of a heterozygous carrier of X-linked retinitis pigmentosa due to mosaicism for a RHO variant

Scientific Reports ◽

10.1038/s41598-020-80400-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ine Strubbe ◽

Caroline Van Cauwenbergh ◽

Julie De Zaeytijd ◽

Sarah De Jaegere ◽

Marieke De Bruyne ◽

...

Keyword(s):

Retinitis Pigmentosa ◽

Somatic Mosaicism ◽

Retinal Disease ◽

Hair Follicles ◽

Disease Genes ◽

Female Carrier ◽

Autofluorescence Imaging ◽

Targeted Next Generation Sequencing ◽

Whole Exome ◽

Next Generation Sequencing Ngs

AbstractWe describe both phenotype and pathogenesis in two male siblings with typical retinitis pigmentosa (RP) and the potentially X-linked RP (XLRP) carrier phenotype in their mother. Two affected sons, two unaffected daughters, and their mother underwent detailed ophthalmological assessments including Goldmann perimetry, color vision testing, multimodal imaging and ISCEV-standard electroretinography. Genetic testing consisted of targeted next-generation sequencing (NGS) of known XLRP genes and whole exome sequencing (WES) of known inherited retinal disease genes (RetNet-WES). Variant validation and segregation analysis were performed by Sanger sequencing. The mutational load of the RHO variant in the mother was assessed in DNA from leucocytes, buccal cells and hair follicles using targeted NGS. Both affected sons showed signs of classical RP, while the mother displayed patches of hyperautofluorescence on blue light autofluorescence imaging and regional, intraretinal, spicular pigmentation, reminiscent of a carrier phenotype of XLRP. XLRP testing was negative. RetNet-WES testing revealed RHO variant c.404G > C p.(Arg135Pro) in a mosaic state (21% of the reads) in the mother and in a heterozygous state in both sons. Targeted NGQSS of the RHO variant in different maternal tissues showed a mutation load between 25.06% and 41.72%. We report for the first time that somatic mosaicism of RHO variant c.404G > C p.(Arg135Pro) mimics the phenotype of a female carrier of XLRP, in combination with heterozygosity for the variant in the two affected sons.

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Assessment of Physico-Chemical and Toxicological Properties of Commercial 2D Boron Nitride Nanopowder and Nanoplatelets

International Journal of Molecular Sciences ◽

10.3390/ijms22020567 ◽

2021 ◽

Vol 22 (2) ◽

pp. 567

Author(s):

Brixhilda Domi ◽

Kapil Bhorkar ◽

Carlos Rumbo ◽

Labrini Sygellou ◽

Spyros N. Yannopoulos ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Boron Nitride ◽

A549 Cells ◽

Reactive Oxygen ◽

Slight Reduction ◽

Oxygen Species ◽

Potential Toxicity ◽

Cellular Models ◽

Physico Chemical ◽

The Impact

Boron nitride (BN) nanomaterials have been increasingly explored for potential applications in chemistry and biology fields (e.g., biomedical, pharmaceutical, and energy industries) due to their unique physico-chemical properties. However, their safe utilization requires a profound knowledge on their potential toxicological and environmental impact. To date, BN nanoparticles have been considered to have a high biocompatibility degree, but in some cases, contradictory results on their potential toxicity have been reported. Therefore, in the present study, we assessed two commercial 2D BN samples, namely BN-nanopowder (BN-PW) and BN-nanoplatelet (BN-PL), with the objective to identify whether distinct physico-chemical features may have an influence on the biological responses of exposed cellular models. Morphological, structural, and composition analyses showed that the most remarkable difference between both commercial samples was the diameter of their disk-like shape, which was of 200–300 nm for BN-PL and 100–150 nm for BN-PW. Their potential toxicity was investigated using adenocarcinomic human alveolar basal epithelial cells (A549 cells) and the unicellular fungus Saccharomycescerevisiae, as human and environmental eukaryotic models respectively, employing in vitro assays. In both cases, cellular viability assays and reactive oxygen species (ROS) determinations where performed. The impact of the selected nanomaterials in the viability of both unicellular models was very low, with only a slight reduction of S. cerevisiae colony forming units being observed after a long exposure period (24 h) to high concentrations (800 mg/L) of both nanomaterials. Similarly, BN-PW and BN-PL showed a low capacity to induce the formation of reactive oxygen species in the studied conditions. Even at the highest concentration and exposure times, no major cytotoxicity indicators were observed in human cells and yeast. The results obtained in the present study provide novel insights into the safety of 2D BN nanomaterials, indicating no significant differences in the toxicological potential of similar commercial products with a distinct lateral size, which showed to be safe products in the concentrations and exposure conditions tested.

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HERVs establish a distinct molecular subtype in stage II/III colorectal cancer with poor outcome

npj Genomic Medicine ◽

10.1038/s41525-021-00177-w ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Mahdi Golkaram ◽

Michael L. Salmans ◽

Shannon Kaplan ◽

Raakhee Vijayaraghavan ◽

Marta Martins ◽

...

Keyword(s):

Colorectal Cancer ◽

Molecular Subtype ◽

Endogenous Retroviruses ◽

Stage Ii ◽

Clinicopathological Factors ◽

Whole Exome ◽

Potential Biomarker ◽

Whole Transcriptome Sequencing ◽

Next Generation Sequencing Ngs ◽

Herv Expression

AbstractColorectal cancer (CRC) is one of the most lethal malignancies. The extreme heterogeneity in survival rate is driving the need for new prognostic biomarkers. Human endogenous retroviruses (hERVs) have been suggested to influence tumor progression, oncogenesis and elicit an immune response. We examined multiple next-generation sequencing (NGS)-derived biomarkers in 114 CRC patients with paired whole-exome and whole-transcriptome sequencing (WES and WTS, respectively). First, we demonstrate that the median expression of hERVs can serve as a potential biomarker for prognosis, relapse, and resistance to chemotherapy in stage II and III CRC. We show that hERV expression and CD8+ tumor-infiltrating T-lymphocytes (TILs) synergistically stratify overall and relapse-free survival (OS and RFS): the median OS of the CD8-/hERV+ subgroup was 29.8 months compared with 37.5 months for other subgroups (HR = 4.4, log-rank P < 0.001). Combing NGS-based biomarkers (hERV/CD8 status) with clinicopathological factors provided a better prediction of patient survival compared to clinicopathological factors alone. Moreover, we explored the association between genomic and transcriptomic features of tumors with high hERV expression and establish this subtype as distinct from previously described consensus molecular subtypes of CRC. Overall, our results underscore a previously unknown role for hERVs in leading to a more aggressive subtype of CRC.

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The Power of Yeast in Modelling Human Nuclear Mutations Associated with Mitochondrial Diseases

Genes ◽

10.3390/genes12020300 ◽

2021 ◽

Vol 12 (2) ◽

pp. 300

Author(s):

Camilla Ceccatelli Berti ◽

Giulia di Punzio ◽

Cristina Dallabona ◽

Enrico Baruffini ◽

Paola Goffrini ◽

...

Keyword(s):

Molecular Mechanisms ◽

Mitochondrial Diseases ◽

Yeast Saccharomyces Cerevisiae ◽

Genome Data ◽

New Genes ◽

Eukaryotic Organism ◽

Novel Variants ◽

Therapeutic Molecules ◽

Nuclear Mutations

The increasing application of next generation sequencing approaches to the analysis of human exome and whole genome data has enabled the identification of novel variants and new genes involved in mitochondrial diseases. The ability of surviving in the absence of oxidative phosphorylation (OXPHOS) and mitochondrial genome makes the yeast Saccharomyces cerevisiae an excellent model system for investigating the role of these new variants in mitochondrial-related conditions and dissecting the molecular mechanisms associated with these diseases. The aim of this review was to highlight the main advantages offered by this model for the study of mitochondrial diseases, from the validation and characterisation of novel mutations to the dissection of the role played by genes in mitochondrial functionality and the discovery of potential therapeutic molecules. The review also provides a summary of the main contributions to the understanding of mitochondrial diseases emerged from the study of this simple eukaryotic organism.

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One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation

Genetics in Medicine ◽

10.1038/s41436-021-01187-w ◽

2021 ◽

Author(s):

Stephen E. Lincoln ◽

Tina Hambuch ◽

Justin M. Zook ◽

Sara L. Bristow ◽

Kathryn Hatchell ◽

...

Keyword(s):

Diagnostic Yield ◽

Copy Number Variants ◽

Low Complexity ◽

Clinical Genetic ◽

Clinical Sensitivity ◽

Pathogenic Variants ◽

Wide Range ◽

Large Indels ◽

Next Generation Sequencing Ngs ◽

The Impact

Abstract Purpose To evaluate the impact of technically challenging variants on the implementation, validation, and diagnostic yield of commonly used clinical genetic tests. Such variants include large indels, small copy-number variants (CNVs), complex alterations, and variants in low-complexity or segmentally duplicated regions. Methods An interlaboratory pilot study used synthetic specimens to assess detection of challenging variant types by various next-generation sequencing (NGS)–based workflows. One well-performing workflow was further validated and used in clinician-ordered testing of more than 450,000 patients. Results In the interlaboratory study, only 2 of 13 challenging variants were detected by all 10 workflows, and just 3 workflows detected all 13. Limitations were also observed among 11 less-challenging indels. In clinical testing, 21.6% of patients carried one or more pathogenic variants, of which 13.8% (17,561) were classified as technically challenging. These variants were of diverse types, affecting 556 of 1,217 genes across hereditary cancer, cardiovascular, neurological, pediatric, reproductive carrier screening, and other indicated tests. Conclusion The analytic and clinical sensitivity of NGS workflows can vary considerably, particularly for prevalent, technically challenging variants. This can have important implications for the design and validation of tests (by laboratories) and the selection of tests (by clinicians) for a wide range of clinical indications.

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