-
Acta Neurochirurgica Feb 2022Malignant peripheral nerve sheath tumor of the vestibulocochlear nerve (VN-MPNST) is exceedingly rare and carries a poor prognosis. Little is known about its underlying...
INTRODUCTION
Malignant peripheral nerve sheath tumor of the vestibulocochlear nerve (VN-MPNST) is exceedingly rare and carries a poor prognosis. Little is known about its underlying genetics and in particular the process of malignant transformation. There is an ongoing debate on whether the transformation is initiated by ionizing radiation. We present here the analysis and comparison of two post-radiation VN-MPNST and one undergoing spontaneous transformation.
METHODS
Four tumors from three patients (radiation-naïve vestibular schwannoma before (VS) and after (VN-MPNST) malignant transformation in addition to two post-radiation VN-MPNST) were subjected to DNA whole-genome microarray and whole-exome sequencing and tumor-specific mutations were called. Mutational signatures were characterized using MuSiCa.
RESULTS
The tumor genomes were characterized predominantly by copy-number aberrations with 36-81% of the genome affected. Even the VS genome was grossly aberrated. The spontaneous malignant transformation was characterized by a near-total whole-genome doubling, disappearance of NF2 mutation and new mutations in three cancer-related genes (GNAQ, FOXO4 and PDGFRB). All tumors had homozygous loss of the tumor suppressor CDKN2A. Neither mutational signature nor copy number profile was associated with ionizing radiation.
CONCLUSION
The VN-MPNST genome in our cases is characterized by large copy-number aberrations and homozygous deletion of CDKN2A. Our study demonstrates a VS with genetic alterations similar to its malignant counterpart, suggesting the existence of premalignant VS. No consistent mutational signature was associated with ionizing radiation.
Topics: Homozygote; Humans; Mutation; Nerve Sheath Neoplasms; Neuroma, Acoustic; Sequence Deletion; Vestibulocochlear Nerve
PubMed: 34816314
DOI: 10.1007/s00701-021-05062-0 -
Nucleic Acids Research Mar 2021The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement....
The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement. Here, we describe paired gRNA deletions along the PIGA locus on the human X chromosome ranging from 17 kb to 2 Mb. We found no compelling linear correlation between deletion size and the deletion efficiency, and there is no substantial impact of topologically associating domains on deletion frequency. Using this precise deletion technique, we have engineered a series of designer deletion cell lines, including one with deletions of two X-chromosomal counterselectable (negative selection) markers, PIGA and HPRT1, and additional cell lines bearing each individual deletion. PIGA encodes a component of the glycosylphosphatidylinositol (GPI) anchor biosynthetic apparatus. The PIGA gene counterselectable marker has unique features, including existing single cell level assays for both function and loss of function of PIGA and the existence of a potent counterselectable agent, proaerolysin, which we use routinely for selection against cells expressing PIGA. These designer cell lines may serve as a general platform with multiple selection markers and may be particularly useful for large scale genome engineering projects such as Genome Project-Write (GP-write).
Topics: Bacterial Toxins; CRISPR-Cas Systems; Cell Engineering; Cell Line; Chromosomes, Human, X; Genetic Markers; Heterozygote; Humans; Membrane Proteins; Mutation; N-Acetylglucosaminyltransferases; Pore Forming Cytotoxic Proteins; RNA; Sequence Deletion
PubMed: 33591308
DOI: 10.1093/nar/gkab035 -
GeroScience Jun 2021Mitochondrial DNA (mtDNA) quality and quantity relate to two hallmarks of aging-genomic instability and mitochondrial dysfunction. Physical performance relies on...
Mitochondrial DNA (mtDNA) quality and quantity relate to two hallmarks of aging-genomic instability and mitochondrial dysfunction. Physical performance relies on mitochondrial integrity and declines with age, yet the interactions between mtDNA quantity, quality, and physical performance are unclear. Using a validated digital PCR assay specific for mtDNA deletions, we tested the hypothesis that skeletal muscle mtDNA deletion mutation frequency (i.e., a measure of mtDNA quality) or mtDNA copy number predicts physical performance in older adults. Total DNA was isolated from vastus lateralis muscle biopsies and used to quantitate mtDNA copy number and mtDNA deletion frequency by digital PCR. The biopsies were obtained from a cross-sectional cohort of 53 adults aged 50 to 86 years. Before the biopsy procedure, physical performance measurements were collected, including VO, modified physical performance test score, 6-min walk distance, gait speed, grip strength, and total lean and leg mass. Linear regression models were used to evaluate the relationships between age, sex, and the outcomes. We found that mtDNA deletion mutation frequency increased exponentially with advancing age. On average from ages 50 to 86, deletion frequency increased from 0.008 to 0.15%, an 18-fold increase. Females may have lower deletion frequencies than males at older ages. We also measured declines in VO and mtDNA copy number with age in both sexes. The mtDNA deletion frequency measured from single skeletal muscle biopsies predicted 13.3% of the variation in VO. Copy number explained 22.6% of the variation in mtDNA deletion frequency and 10.4% of the lean mass variation. We found predictive relationships between age, mtDNA deletion mutation frequency, mtDNA copy number, and physical performance. These data are consistent with a role for mitochondrial function and genome integrity in maintaining physical performance with age. Analyses of mtDNA quality and quantity in larger cohorts and longitudinal studies could extend our understanding of the importance of mitochondrial DNA in human aging and longevity.
Topics: Aged; Aged, 80 and over; Cross-Sectional Studies; DNA Copy Number Variations; DNA, Mitochondrial; Female; Humans; Male; Middle Aged; Mitochondria; Muscle, Skeletal; Physical Functional Performance; Sequence Deletion
PubMed: 33740224
DOI: 10.1007/s11357-021-00351-z -
Plant Cell Reports Sep 2023The deletion mutated rpoC2 leads to yellow stripes of Clivia miniata var. variegata by down regulating the transcription of 28 chloroplast genes and disturbing...
The deletion mutated rpoC2 leads to yellow stripes of Clivia miniata var. variegata by down regulating the transcription of 28 chloroplast genes and disturbing chloroplast biogenesis and thylakoid membrane development. Clivia miniata var. variegata (Cmvv) is a common mutant of Clivia miniata but its genetic basis is unclear. Here, we found that a 425 bp deletion mutation of chloroplast rpoC2 underlies the yellow stripes (YSs) of Cmvv. Both RNA polymerase PEP and NEP coexist in seed-plant chloroplasts and the β″ subunit of PEP is encoded by rpoC2. The rpoC2 mutation changed the discontinuous cleft domain required to form the PEP central cleft for DNA binding from 1103 to 59 aa. RNA-Seq revealed that 28 chloroplast genes (cpDEGs) were all down-regulated in YSs, of which, four involved in chloroplast protein translation and 21 of photosynthesis system (PS)I, PSII, cytochrome b/f complex and ATP synthase are crucial for chloroplast biogenesis/development. The accuracy and reliability of RNA-Seq was verified by qRT-PCR. Moreover, the chlorophyll (Chl) a/b content, ratio of Chla/Chlb and photosynthetic rate (Pn) of YS decreased significantly. Meanwhile, chloroplasts of the YS mesophyll cells were smaller, irregular in shape, contain almost no thylakoid membrane, and even proplastid was found in YS. These findings indicate that the rpoC2 mutation down-regulated expression of the 28 cpDEGs, which disturb chloroplast biogenesis and its thylakoid membrane development. Thus, there are not enough PSI and II components to bind Chl, so that the corresponding areas of the leaf are yellow and show a low Pn. In this study, the molecular mechanism of three phenotypes of F (Cmvv ♀ × C. miniata ♂) was revealed, which lays a foundation for the breeding of variegated plants.
Topics: Reproducibility of Results; Plant Breeding; Chloroplasts; Mutation; Plant Leaves; Amaryllidaceae; Sequence Deletion; Gene Expression Regulation, Plant
PubMed: 37326841
DOI: 10.1007/s00299-023-03039-0 -
Brain Tumor Pathology Jan 2024Deletion of CDKN2A occurs in 50% of glioblastomas (GBM), and IFNA locus deletion in 25%. These genes reside closely on chromosome 9. We investigated whether CDKN2A and...
Deletion of CDKN2A occurs in 50% of glioblastomas (GBM), and IFNA locus deletion in 25%. These genes reside closely on chromosome 9. We investigated whether CDKN2A and IFNA were co-deleted within the same heterogeneous tumour and their prognostic implications. We assessed CDKN2A and IFNA14 deletions in 45 glioma samples using an in-house three-colour FISH probe. We examined the correlation between p16 protein expression (via IHC) and CDKN2A deletion along with the impact of these genomic events on patient survival. FISH analyses demonstrated that grades II and III had either wildtype (wt) or amplified CDKN2A/IFNA14, whilst 44% of GBMs harboured homozygous deletions of both genes. Cores with CDKN2A homozygous deletion (n = 11) were negative for p16. Twenty p16 positive samples lacked CDKN2A deletion with some of cells showing negative p16. There was heterogeneity in IFNA14/CDKN2A ploidy within each GBM. Survival analyses of primary GBMs suggested a positive association between increased p16 and longer survival; this persisted when considering CDKN2A/IFNA14 status. Furthermore, wt (intact) CDKN2A/IFNA14 were found to be associated with longer survival in recurrent GBMs. Our data suggest that co-deletion of CDKN2A/IFNA14 in GBM negatively correlates with survival and CDKN2A-wt status correlated with longer survival, and with second surgery, itself a marker for improved patient outcomes.
Topics: Humans; Cyclin-Dependent Kinase Inhibitor p16; Gene Deletion; Glioblastoma; Homozygote; Sequence Deletion
PubMed: 38097874
DOI: 10.1007/s10014-023-00473-6 -
Cancer Research Communications Apr 2023Gastrointestinal stromal tumors (GIST) with exon 11 deletions involving in codons 557-558 ( Δ557-558) exhibit higher proliferation rates and shorter disease-free...
UNLABELLED
Gastrointestinal stromal tumors (GIST) with exon 11 deletions involving in codons 557-558 ( Δ557-558) exhibit higher proliferation rates and shorter disease-free survival times compared with GISTs with other exon 11 mutations. We analyzed 30 GIST cases and observed genomic instability and global DNA hypomethylation only in high-risk malignant GISTs with Δ557-558. Whole-genome sequencing revealed that the high-risk malignant GISTs with Δ557-558 (12 cases) had more structural variations (SV), single-nucleotide variants, and insertions and deletions compared with the low-risk, less malignant GISTs with Δ557-558 (six cases) and the high-risk (six cases) or low-risk (6 cases) GISTs with other exon 11 mutations. The malignant GISTs with Δ557-558 showed higher frequency and significance in copy number (CN) reduction on chromosome arms 9p and 22q, and 50% of them had LOH or CN-dependent expression reduction in . In addition, SVs with driver potential were detected in 75% of them, in which and were recurrently identified. Genome-wide DNA methylation and gene expression analyses showed global intergenic DNA hypomethylation, upregulation, and higher expression signatures, including p53 inactivation and chromosomal instability, as characteristics of malignant GISTs with Δ557-558 that distinguished them from other GISTs. These genomic and epigenomic profiling results revealed that Δ557-558 mutations are associated with increased genomic instability in malignant GISTs.
SIGNIFICANCE
We present genomic and epigenomic insights into the malignant progression of GISTs with exon 11 deletions involving in 557-558, demonstrating their unique chromosomal instability and global intergenic DNA hypomethylation.
Topics: Humans; DNA, Intergenic; Epigenomics; Exons; Gastrointestinal Stromal Tumors; Genomic Instability; Sequence Deletion
PubMed: 37377752
DOI: 10.1158/2767-9764.CRC-22-0364 -
Molecular Genetics & Genomic Medicine May 2023Pseudohypoparathyroidism (PHP) is a series of diseases related to pathological changes and neurocognitive and endocrine abnormalities, mainly due to the GNAS mutation on...
BACKGROUND
Pseudohypoparathyroidism (PHP) is a series of diseases related to pathological changes and neurocognitive and endocrine abnormalities, mainly due to the GNAS mutation on chromosome 20q13.2, which weakens receptor-mediated hormone signal transduction. Considering its complex genetic and epigenetic characteristics, GNAS may produce complex clinical phenotypes in families or sporadic cases. This study presented a case of familial PHP caused by a deletion mutation in the 20q13.2 region.
METHODS AND RESULTS
The proband and her second daughter had PHP, and the proband's mother had pseudo-PHP. Whole-genome sequencing revealed that the proband had an 849.81 kb deletion spanning GNAS near the maternal 20q13.2 chromosome. Multiplex ligation-dependent probe amplification methylation analysis indicated that the proband as well as her mother and second daughter had seemingly abnormal GNAS methylation. This is different from the phenotype (feeding difficulty, slow growth, and special facial features) of previously reported cases with the deletion of fragments near the 20q13.2 chromosome.
CONCLUSIONS
This report demonstrated the variability of 20q13.2 deletion phenotypes and the clinical importance of using multiple molecular genetic detection methods.
Topics: Female; Humans; GTP-Binding Protein alpha Subunits, Gs; DNA Methylation; Chromogranins; Pseudohypoparathyroidism; Sequence Deletion
PubMed: 36669868
DOI: 10.1002/mgg3.2144 -
European Journal of Endocrinology Dec 2023
Topics: Humans; Cohort Studies; Retrospective Studies; Sequence Deletion; DNA, Mitochondrial
PubMed: 38039503
DOI: 10.1093/ejendo/lvad165 -
Virus Research Jan 2022Synthetic biology has been applied countless times for the modification and improvement of bacterial strains and for the synthesis of products that do not exist in...
Synthetic biology has been applied countless times for the modification and improvement of bacterial strains and for the synthesis of products that do not exist in nature. Phages are natural predators of bacteria controlling their population levels; however, their genomes carry several genes with unknown functions. In this work, Bacteriophage Recombineering of Electroporated DNA was used to assess the influence of deletion of a single gene with unknown function in the overall replication parameters of Salmonella phage PVP-SE2. Deletion of ORF_01, transcribed immediately after infection, reduced both the latent and rise periods by 5 min in PVP-SE2ΔORF_01 compared to the wild-type phage. A direct consequence of the deletion led to a smaller progeny release per infected cell by the mutant compared to the wild-type phage. Despite the difference in growth characteristics, the mutant phage remained infective towards exponentially growing cells. The mutation engineered endured for at least ten passages, showing that there is no reversion back to the wild-type sequence. This study provides proof of concept that methodologies used for phage engineering should always be complemented by phage growth characterization to assess whether a mutation can trigger undesirable characteristics.
Topics: Bacteriophages; Gene Deletion; Salmonella Phages; Salmonella enteritidis; Sequence Analysis, DNA
PubMed: 34902446
DOI: 10.1016/j.virusres.2021.198654 -
Molecular Reproduction and Development Jul 2021Glands of the uterus are essential for the establishment of pregnancy in mice and their products regulate embryo implantation and stromal cell decidualization critical...
Glands of the uterus are essential for the establishment of pregnancy in mice and their products regulate embryo implantation and stromal cell decidualization critical for pregnancy establishment. Forkhead box A2 (FOXA2) is expressed specifically in the glands and a critical regulator of their differentiation, development and function. Progesterone and FOXA2 regulate members of a serine proteinase gene family (Prss28 and Prss29). Here, CRISPR-Cas9 genome-editing was used to create mice with a heterozygous or homozygous deletion of Prss28 or/and Prss29 to determine their biological roles in uterine function. Female mice lacking Prss28 and Prss29 or both developed normally and were fertile without alterations in uterine histoarchitecture, uterine gland number, or and gene expression. Thus, Prss28 and Prss29 are dispensable for female fertility and do not impact endometrial gland development or uterine function mice.
Topics: Animals; CRISPR-Cas Systems; Endometrium; Female; Gene Editing; Gene Knockout Techniques; Male; Mice; Mice, Knockout; Pregnancy; Sequence Deletion; Serine Endopeptidases; Uterus
PubMed: 33973295
DOI: 10.1002/mrd.23473