Mutations In Somatic Cells Research Articles

Overcoming treatment challenges in myelofibrosis and polycythemia vera: the role of ruxolitinib.

Myelofibrosis (MF) and polycythemia vera (PV) are BCR-ABL1-negative myeloproliferative neoplasms associated with somatic hematopoietic stem cell mutations leading to over activation of JAK–STAT signaling. MF and PV are pathogenically related and share specific clinical features such as splenomegaly and constitutional symptoms. The MF phenotype is dominated by the effects of progressive bone marrow fibrosis resulting in shortened survival. In contrast, elevated thrombosis risk due to erythrocytosis is the primary clinical concern in PV. Ruxolitinib, an oral JAK1/JAK2 inhibitor, is approved in the USA for the treatment of patients with intermediate- or high-risk MF and patients with PV who have had an inadequate response to or are intolerant of hydroxyurea. For MF, results of two phase III studies demonstrated that ruxolitinib therapy reduced spleen volume and MF-related symptom burden, improved quality-of-life measures, and was associated with prolonged overall survival. Treatment benefits were generally sustained with continued therapy. Dose-dependent cytopenias were common but generally manageable with transfusions (for anemia), dose reduction, or treatment interruption. Optimal dosing management is critical to maintain long-term treatment benefit, because cessation of therapy resulted in rapid return of symptoms to baseline levels. Results of the phase III PV trial showed that ruxolitinib was significantly more effective than standard therapy in controlling hematocrit levels and improving splenomegaly and PV-related symptoms. Only 1 of 110 patients in the ruxolitinib arm compared with 6 of 112 patients in the control arm experienced a thromboembolic event through week 32. Grade ≥3 cytopenias were uncommon.

Efficient Generation of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair.

Precise genome editing in livestock is of great value for the fundamental investigation of disease modeling. However, genetically modified pigs carrying subtle point mutations were still seldom reported despite the rapid development of programmable endonucleases. Here, we attempt to investigate single-stranded oligonucleotides (ssODN) mediated knockin by introducing two orthologous pathogenic mutations, p.E693G for Alzheimer's disease and p.G2019S for Parkinson's disease, into porcine APP and LRRK2 loci, respectively. Desirable homology-directed repair (HDR) efficiency was achieved in porcine fetal fibroblasts (PFFs) by optimizing the dosage and length of ssODN templates. Interestingly, incomplete HDR alleles harboring partial point mutations were observed in single-cell colonies, which indicate the complex mechanism of ssODN-mediated HDR. The effect of mutation-to-cut distance on incorporation rate was further analyzed by deep sequencing. We demonstrated that a mutation-to-cut distance of 11 bp resulted in a remarkable difference in HDR efficiency between two point mutations. Finally, we successfully obtained one cloned piglet harboring the orthologous p.C313Y mutation at the MSTN locus via somatic cell nuclear transfer (SCNT). Our proof-of-concept study demonstrated efficient ssODN-mediated incorporation of pathogenic point mutations in porcine somatic cells, thus facilitating further development of disease modeling and genetic breeding in pigs.

Genotoxicity and antigenotoxicity study of aqueous and hydro-methanol extracts of Spondias mombin L., Nymphaea lotus L. and Luffa cylindrical L. using animal bioassays.

Spondias mombin (Linn), Nymphaea lotus (Linn) and Luffa cylindrica (Linn) (syn Luffa aegyptiaca Mill) are plants traditionally used as food ingredients and in the management of diseases, including cancer, in Nigeria. Despite the therapeutic potentials attributed to these plants, reports on their genotoxicity are scanty. In this study, the genotoxicity of the aqueous and hydro-methanol extract of these plants was evaluated using mouse bone marrow micronucleus and sperm morphology assays. Antigenotoxicity was assessed by the bone marrow micronucleus test. The highest attainable dose of 5 000 mg/kg according to OECD guidelines was first used to assess acute toxicity of the aqueous and hydro-methanol extracts in Swiss albino mice. For each extract, there were five groups of mice (n=4/group) treated with different concentrations of the extract as against the negative and positive control group for the genotoxicity study. In the antigenotoxicity study, five groups of mice were exposed to five different concentrations of the extracts along with 60 mg/kg of methyl methane sulfonate (MMS), which was used to induce genotoxicity. The mice were administered 0.2 mL of extract per day for 10 days in the genotoxicity and antigenotoxicity groups. Administration of each of the extracts at the concentration of 5 000 mg/kg did not induce acute toxicity in mice. At the concentrations tested, all the extracts, except aqueous S. mombin, increased micronucleated polychromatic erythrocytes. The aqueous and hydro-methanol extracts of N. lotus increased the frequency of aberrant sperm cells. All the extracts were also able to ameliorate MMS induced genotoxicity in bone marrow cells of the exposed mice. The results showed the potential of the extracts to induce somatic and germ cell mutation in male mice. The extracts also ameliorated the genotoxic effect of MMS.

Somatic-cell mutation induced by short exposures to cigarette smoke in urate-null, oxidative stress-sensitive Drosophila

We previously reported that a urate-null strain of Drosophila is hypersensitive to cigarette smoke (CS), and we suggested that CS induces oxidative stress in Drosophila because uric acid is a potent antioxidant. Although the carcinogenic risk of CS exposure is widely recognized; documentation of in vivo genotoxic activity of environmental CS, especially gaseous-phase CS, remains inconclusive. To date, somatic-cell mutations in Drosophila resulting from exposure to CS have not been detected via the somatic mutation and recombination test (wing spot test) with wild-type flies, a widely used Drosophila assay for the detection of somatic-cell mutation; moreover, genotoxicity has not been documented via a DNA repair test that involves DNA repair-deficient Drosophila. In this study, we used a new Drosophila strain (y v ma-l; mwh) to examine the mutagenicity induced by gaseous-phase CS; these flies are urate-null due to a mutation in ma-l, and they are heterozygous for multiple wing hair (mwh), a mutation that functions as a marker for somatic-cell mutation. In an assay with this newly developed strain, a superoxide anion-producing weed-killer, paraquat, exhibited significant mutagenicity; in contrast, paraquat was hardly mutagenic with a wild-type strain. Drosophila larvae were exposed to CS for 2, 4 or 6h, and then kept at 25°C on instant medium until adulthood. After eclosion, mutant spots, which consisted of mutant hairs on wings, were scored. The number of mutant spots increased significantly in an exposure time-dependent manner in the urate-null females (ma-l (-/-)), but not in the urate-positive females (ma-l (+/-)). In this study, we showed that short-term exposure to CS was mutagenic in this in vivo system. In addition, we obtained suggestive data regarding reactive oxygen species production in larva after CS exposure using the fluorescence probe H2DCFDA. These results suggest that oxidative damage, which might be countered by uric acid, was partly responsible for induction of somatic cell mutations in Drosophila larvae exposed to CS.

More than one genotype: how common is intracolonial genetic variability in scleractinian corals?

In recent years, a few colonial marine invertebrates have shown intracolonial genetic variability, a previously unreported phenomenon. Intracolonial genetic variability describes the occurrence of more than a single genotype within an individual colony. This variability can be traced back to two underlying processes: chimerism and mosaicism. Chimerism is the fusion of two or more individuals, whereas mosaicism mostly derives from somatic cell mutations. Until now, it remained unclear to what degree the ecologically important group of hermatypic (reef building) corals might be affected. We investigate the occurrence of intracolonial genetic variability in five scleractinian corals: Acropora florida, Acropora hyacinthus, Acropora sarmentosa, Pocillopora species complex and Porites australiensis. The main focus was to test different genera for the phenomenon via microsatellite markers and to distinguish which underlying process caused the genetic heterogeneity. Our results show that intracolonial genetic variability was common (between 46.6% for A.sarmentosa and 23.8% for P.species complex) in all tested corals. The main process was mosaicism (69 cases of 222 tested colonies), but at least one chimera existed in every species. This suggests that intracolonial genetic variability is widespread in scleractinian corals and could challenge the view of a coral colony as an individual and therefore a unit of selection. However, it might also hold potential for colony survival under rapidly changing environmental conditions.

Polymorphisms in rs 13181 of ERCC2 gene in the personnel of Stepnogorsk gorno chemiches kombinat, Kazakhstan

The possibility of mutations in human somatic cells induced by action of radiation in low doses is a subject of investigations and discussion. The protein encoded by ERCC2 (XPD) gene is involved in transcription‐coupled nucleotide excision repair and is associated with some types of cancer. The aim of this study was to determine the influence of chronic action of radiation in low doses to the level of the single nucleotide polymorphisms in DNA of the personnel of uranium enrichment plant, Akmola, Kazakhstan. The sample set consisted of 234 (52–Kazakhs, 182–Russians) men workers of and the control group includes 289 (129–Kazakhs, 160–Russians). Genotyping was performed by restriction fragments length polymorphism analysis of PCR products of the rs13181 site (T/G) of (T/G) of ERCC2 gene using PstI. In the case‐control studythe allele frequencies and genotypes distribution were compared in the groups of personnel and controlsusing STATISTICA v. 5.0 software.The differences in allele frequencies and genotypes distribution in the groups of personnel and controls were evaluated neither in Kazakh nor in Russian groups. At the next stage comparing of allele frequencies and genotypes distribution was performed in the subgroups of the workers divided according accumulated dose: less than 40 mZv, 40‐100 mZv.The differences were found in genotype distribution only in the group of 50 Russian workers with accumulated dose 40–100 mZv compared with 100 of controls: χ2=6.796, p=0.033, OR=1.363, 95%CI=0.844‐2.201. The group of persons who accumulated dose more than 100 mZv encountered only 5 persons and was not included in calculations.

Confirmation of Pig-a mutation in flow cytometry-identified CD48-deficient T-lymphocytes from F344 rats.

The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene.

Evidence that mutation accumulation does not cause aging in Saccharomyces cerevisiae.

The concept that mutations cause aging phenotypes could not be directly tested previously due to inability to identify age-related mutations in somatic cells and determine their impact on organismal aging. Here, we subjected Saccharomyces cerevisiae to multiple rounds of replicative aging and assessed de novo mutations in daughters of mothers of different age. Mutations did increase with age, but their low numbers, < 1 per lifespan, excluded their causal role in aging. Structural genome changes also had no role. A mutant lacking thiol peroxidases had the mutation rate well above that of wild-type cells, but this did not correspond to the aging pattern, as old wild-type cells with few or no mutations were dying, whereas young mutant cells with many more mutations continued dividing. In addition, wild-type cells lost mitochondrial DNA during aging, whereas shorter-lived mutant cells preserved it, excluding a causal role of mitochondrial mutations in aging. Thus, DNA mutations do not cause aging in yeast. These findings may apply to other damage types, suggesting a causal role of cumulative damage, as opposed to individual damage types, in organismal aging.

Cell-of-origin chromatin organization shapes the mutational landscape of cancer.

Cancer is a disease potentiated by mutations in somatic cells. Cancer mutations are not distributed uniformly along the genome. Instead, different genomic regions vary by up to 5-fold in the local density of somatic mutations1, posing a fundamental problem for statistical methods of cancer genomics. Epigenomic organization has been proposed as a major determinant of the cancer mutational landscape1-5. However, both somatic mutagenesis and epigenomic features are highly cell-type-specific6,7. We investigated the distribution of mutations in multiple samples of diverse cancer types and compared them to cell-type-specific epigenomic features. Here, we show that chromatin accessibility and modification, together with replication timing, explain up to 86% of the variance in mutation rates along cancer genomes. Overwhelmingly, the best predictors of local somatic mutation density are epigenomic features derived from the most likely cell type of origin of the corresponding malignancy. Moreover, we find that cell-of-origin chromatin features are much stronger determinants of cancer mutation profiles than chromatin features of cognate cancer cell lines. We show further that the cell type of origin of a cancer can be accurately determined based on the distribution of mutations along its genome. Thus, DNA sequence of a cancer genome encompasses a wealth of information about the identity and epigenomic features of its cell of origin.

Protection of the gametes embryo/fetus from prenatal radiation exposure.

There is no convincing evidence of germline mutation manifest as heritable disease in the offspring of humans attributable to ionizing radiation, yet radiation clearly induces mutations in microbes and somatic cells of rodents and humans. Doses to the embryo estimated to be in the range of 0.15-0.2 Gy during the pre-implantation and pre-somite stages may increase the risk of embryonic loss. However, an increased risk of congenital malformations or growth retardation has not been observed in the surviving embryos. These results are primarily derived from mammalian animal studies and are referred to as the "all-or-none phenomenon." The tissue reaction effects of ionizing radiation (previously referred to as deterministic effects) are congenital malformations, mental retardation, decreased intelligence quotient, microcephaly, neurobehavioral effects, convulsive disorders, growth retardation (height and weight), and embryonic and fetal death (miscarriage, stillbirth). All these effects are consistent with having a threshold dose below which there is no increased risk. The risk of cancer in offspring that have been exposed to diagnostic x-ray procedures while in utero has been debated for 55 y. High doses to the embryo or fetus (e.g., >0.5 Gy) increase the risk of cancer. Most pregnant women exposed to x-ray procedures and other forms of ionizing radiation today received doses to the embryo or fetus <0.1 Gy. The risk of cancer in offspring exposed in utero at exposures <0.1 Gy is controversial and has not been fully resolved. Diagnostic imaging procedures using ionizing radiation that are clinically indicated for the pregnant patient and her fetus should be performed because the clinical benefits outweigh the potential oncogenic risks.

Tracking and transforming neocortical progenitors by CRISPR/Cas9 gene targeting and PiggyBac transposase lineage labeling

The ability to induce targeted mutations in somatic cells in developing organisms and then track the fates of those cells is a powerful approach both for studying neural development and for modeling human disease. The CRISPR/Cas9 system allows for such targeted mutagenesis, and we therefore tested it in combination with a piggyBac transposase lineage labeling system to track the development of neocortical neural progenitors with targeted mutations in genes linked to neurodevelopmental disruptions and tumor formation. We show that sgRNAs designed to target PTEN successfully decreased PTEN expression, and led to neuronal hypertrophy and altered neuronal excitability. Targeting NF1, by contrast, caused increased astrocytogenesis at the expense of neurogenesis, and combined targeting of three tumor suppressors (PTEN, NF1 and P53) resulted in formation of glioblastoma tumors. Our results demonstrate that CRISPR/Cas9 combined with piggyBac transposase lineage labeling can produce unique models of neurodevelopmental disruption and tumors caused by somatic mutation in neural progenitors.

Intraclonal Heterogeneity Associates with Clonal Stability in Multiple Myeloma

Multiple myeloma (MM) pathogenesis has been explained for many years by the cancer biology dogma introduced by Peter Nowell: first, a single plasma cell would be immortalized by an error in the immunoglobulin genes rearrangement process; then, a progressive stepwise acquisition of somatic cell mutations would induce a sequential selection and domination by the fittest clone. In line with this idea of “myeloma stability”, SNP arrays studies in diagnostic-relapse paired samples have revealed the presence of common clonal characteristics. Biologically, the M-protein remains usually constant across MM evolution and further, the variable domain of the rearranged immunoglobulin heavy chain genes (or CDR3 region) has been used as a patient-specific myeloma fingerprint in minimal residual disease (MRD) studies. However, massive genome studies with Next Generation Sequencing (NGS) have challenged this concept, showing a significant intraclonal heterogeneity at diagnosis with the possible presence of several clonal progenitors or tumor-initiating cells.In this study, we have characterized and compared the CDR3 region in 52-paired samples from 26 MM patients aiming: 1) to assess mono-clonality in MM evolution through the analysis of the CDR3 sequence and, 2) to validate ASO RQ-PCR approaches for MRD in MM, based on the constancy and specificity of the CDR3 region.Samples were obtained at diagnosis and progression (19 pairs) or at 2 different timepoints of progressive disease (7 pairs). Median time between sampling was 2 years. M-protein subtype remained stable in all pairs but 1, associated with a light-chain escape phenomenon. All samples proceeded from bone marrow (BM) except for 2 pairs, composed by BM and extramedullary disease (spleen and testes). Two major cytogenetic changes were identified: increased 13q14 deletion (from 7 to 54%) in 1 pair and increased 17p (p53) deletion (from 5 to 87%) in a further one. Treatments administered between sampling included most of the current approaches used in MM (data not shown).Genomic DNA isolation, PCR amplification and sequencing were performed following conventional methods. Germline VH, DH and JH segments were identified by comparison with public databases. CDR3 region was first identified in all samples and then compared between the two samples in the 26 pairs: the sequence of nucleotides was constantly identical in each pair, including those associated with major cytogenetic changes, a light-chain escape, extramedullar vs. BM infiltration and relapsed (and therefore, treatment selected) vs. refractory disease. Therefore, we can first conclude that the main tumor clone in MM retains a specific signature across all stages of disease evolution that allows the identification of samples as evolutionary related. This major clone signature is not modified by clinical or biological changes in the disease nor under different treatment pressures and would thus identify disease relapse and progression.Our results have also a clear impact on the validity of molecular MRD techniques. The high rate of complete responses (up to 50-60%) currently achieved in MM has prompted the use of new techniques for disease assessment. Today, ASO RQ-PCR, based on the use of specific primers and probes complementary of the VDJH rearrangement, continues to be the most sensitive approach. One pitfall of this technique would be the potential instability of PCR targets over time, which would induce false negative results. In B-cell precursor ALL, this is estimated to happen in 30-40% of cases but has not been deeply evaluated in MM yet. With the present study, we can also conclude that the junction region of the VDJH rearrangement in MM constantly identifies the myeloma cells responsible for relapse and therefore can be used as a reliable target for MRD assessment by ASO RQ-PCR and more recently, by NGS methods.If the CDR3 region remains stable, the novel concept of clonal tiding in MM should not be interpreted as a poly- or oligoclonal but subclonal. In MM, tides can be subclonal, but the ocean remains monoclonal. DisclosuresNo relevant conflicts of interest to declare.

Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.

The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)—three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C—and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.

Somatic cell mutations caused by 365 nm LED-UVA due to DNA double-strand breaks through oxidative damage.

Evidence is accumulating indicating that UVA (320-400 nm ultraviolet light) plays an important role in photo-carcinogenesis. UVA is thought to produce reactive oxygen species in irradiated cells through photo-activation of inherent photosensitizers, and was recently reported to cause DNA double-strand breaks (DSBs) in exposed cells. We have investigated the involvement of UVA in mutations and DNA damage in somatic cells using Drosophila melanogaster larvae. Using the Okazaki Large Spectrograph, we previously observed that longer wavelength UVA (>330 nm) was more mutagenic in post-replication repair-deficient D. melanogaster (mei-41) than in the nucleotide excision repair-deficient strain (mei-9). LED-light has recently been developed as a high-dose-rate UVA source. LED-UVA light (365 nm) was also more mutagenic in mei-41 than in mei-9. The mei-41 gene was shown to be an orthologue of the human ATR gene, which is involved in the repair of DSBs through phosphorylation of histone H2AX. In order to estimate the extent to which oxidative damage contributes to mutation, we established a new D. melanogaster strain (urate-null mutant) that is sensitive to oxidative damage and has a marker to detect somatic cell mutations. When somatic cell mutations were examined using this strain, LED-UVA was mutagenic in the urate-null strain at doses that were non-mutagenic in the urate-positive strain. In an effort to investigate the generation of DSBs, we examined the presence of phosphorylated histone H2AvD (H2AX D. melanogaster homologue). At high doses of LED-UVA (>800 kJ m(-2)), levels of phosphorylated H2AvD (γ-H2AvD) increased significantly in the urate-null strain. Moreover, the level of γ-H2AvD increased in the excision repair-deficient strain but not in the ATR-deficient strain following UVA-irradiation. These results supported the notion that the generation of γ-H2AvD was mediated by the function of the mei-41 gene. It was reported that ATR functions on DSB repair in D. melanogaster. Taken together, we propose a possible pathway for UVA-induced mutation, whereby DNA double-strand breaks resulting from oxidative stress might be responsible for UVA-induced mutation in somatic cells of D. melanogaster larvae.

Fibroblast growth factor receptors: multifactorial-contributors to tumor initiation and progression.

Fibroblast growth factor receptors (FGFRs), encoded by four genes (FGFR1, FGFR2, FGFR3, and FGFR4) are tightly associated with many biological processes such as organ development, cell proliferation and migration. Studies over the past decades have validated the pivotal roles FGFRs play in tumorigenesis due to the regulation of diverse tumorigenesis-related processes, including cell survival, proliferation, inflammation, metastasis and angiogenesis. Interestingly, FGFR mutations in somatic cells leading to tumorigenesis and those in germ cells leading to developmental disorders are identical, suggesting that FGFR mutations result in different diseases due to their spatio-temporal expression. Thus, discoveries in developmental biology may also be applicable to cancer. FGFRs regulate the expression and/or the activity of a myriad of molecules (e.g. matrix metalloproteinases (MMPs) and Snail) that are tightly linked to tumorigenesis by four main signaling pathways (RAS-MAPK, PI3K-AKT, PLCγ-PIP2, and STAT), as well as other minor branches. Epigenetic and genetic alteration of FGFR genes, including DNA methylation, histone remodeling, microRNA regulation, single nucleotide polymorphisms (SNPs), gene missense mutations, amplification, and fusion of FGFRs with other genes, which result in gain or loss of FGFR function, have been identified in many types of cancer. In this review, we focus in particular on recent advances in the relationship between FGFR disorders and tumorigenesis.

Correlation study on adiponectin gene SNP45 and long-term oxidative stress in patients with diabetes and carotid atherosclerosis.

The aim of the present study was to investigate the correlation between the adiponectin gene single nucleotide polymorphism (SNP)45 T/G and long-term oxidative stress in type II diabetes mellitus (T2DM) patients with carotid atherosclerosis. Patients with T2DM were divided into non-carotid atherosclerosis and carotid atherosclerosis groups, which were then subsequently divided into TT and TG + GG groups according to the adiponectin SNP45 T/G genotypes. Enzyme-linked immunosorbent assay, TaqMan probe quantitative polymerase chain reaction (PCR), PCR-TaqMan, color Doppler and other methods were used to determine the adiponectin levels, gene polymorphisms, acquired mitochondrial DNA (mtDNA) A3243G somatic cell mutation rates and the carotid intima-media thickness. The somatic cell mutation rate of acquired mtDNA A3243A/G in the T2DM carotid atherosclerosis group was significantly higher compared with the group without carotid atherosclerosis. In addition, the acquired mtDNA A3243A/G somatic cell mutation rate in the T2DM carotid atherosclerosis group with the adiponectin gene SNP45 TT genotype was significantly lower compared with the SNP45 TG/GG genotype group. T2DM combined with carotid atherosclerosis was associated with long-term oxidative stress. In addition, adiponectin gene SNP45 T/G was associated with increased mtDNA A3243A/G somatic mutation rates in T2DM patients with carotid atherosclerosis. Therefore, adiponectin gene polymorphisms may lead to diabetes atherosclerosis through oxidative stress.

Somatic Correction of Junctional Epidermolysis Bullosa by a Highly Recombinogenic AAV Variant

Definitive correction of disease causing mutations in somatic cells by homologous recombination (HR) is an attractive therapeutic approach for the treatment of genetic diseases. However, HR-based somatic gene therapy is limited by the low efficiency of gene targeting in mammalian cells and replicative senescence of primary cells ex vivo, forcing investigators to explore alternative strategies such as retro- and lentiviral gene transfer, or genome editing in induced pluripotent stem cells. Here, we report correction of mutations at the LAMA3 locus in primary keratinocytes derived from a patient affected by recessive inherited Herlitz junctional epidermolysis bullosa (H-JEB) disorder using recombinant adenoassociated virus (rAAV)-mediated HR. We identified a highly recombinogenic AAV serotype, AAV-DJ, that mediates efficient gene targeting in keratinocytes at clinically relevant frequencies with a low rate of random integration. Targeted H-JEB patient cells were selected based on restoration of adhesion phenotype, which eliminated the need for foreign sequences in repaired cells, enhancing the clinical use and safety profile of our approach. Corrected pools of primary cells assembled functional laminin-332 heterotrimer and fully reversed the blistering phenotype both in vitro and in skin grafts. The efficient targeting of the LAMA3 locus by AAV-DJ using phenotypic selection, together with the observed low frequency of off-target events, makes AAV-DJ based somatic cell targeting a promising strategy for ex vivo therapy for this severe and often lethal epithelial disorder.

One-step generation of knockout pigs by zygote injection of CRISPR/Cas system

The pig is an important livestock for food supply and an ideal model for various human diseases. Efficient and precise genetic engineering in pigs holds great promise in agriculture and biomedicine1. Using currently available approach, generating specific gene modifications in pigs requires two steps. First, site-specific nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are used to generate targeted mutations in pig somatic cells. Then the engineered somatic nucleus is used to generate cloned animals using somatic cell nuclear transfer (SCNT) technology2,3. The complex design and generation of ZFNs and TALENs, as well as the technical challenges of SCNT, greatly limit the application of this method.

Recognizing Paradigm Instability in Theories of Carcinogenesis

The dominant view that cancer arises from successive mutations in somatic cells is so unquestioned that accumulating challenges from experimental and observational results are rarely addressed in the scientific literature. Based on these challenges, we argue that the scientific understanding of carcinogenesis has entered a period of paradigm instability. New research directions are therefore needed.

Return of Secondary Genomic Findings vs Patient Autonomy

Return of Secondary Genomic Findings vs Patient Autonomy