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Science (New York, N.Y.) Oct 2022The predominant mutation causing cystic fibrosis, a deletion of phenylalanine 508 (Δ508) in the cystic fibrosis transmembrane conductance regulator (CFTR), leads to...
The predominant mutation causing cystic fibrosis, a deletion of phenylalanine 508 (Δ508) in the cystic fibrosis transmembrane conductance regulator (CFTR), leads to severe defects in CFTR biogenesis and function. The advanced therapy Trikafta combines the folding corrector tezacaftor (VX-661), the channel potentiator ivacaftor (VX-770), and the dual-function modulator elexacaftor (VX-445). However, it is unclear how elexacaftor exerts its effects, in part because the structure of Δ508 CFTR is unknown. Here, we present cryo-electron microscopy structures of Δ508 CFTR in the absence and presence of CFTR modulators. When used alone, elexacaftor partially rectified interdomain assembly defects in Δ508 CFTR, but when combined with a type I corrector, did so fully. These data illustrate how the different modulators in Trikafta synergistically rescue Δ508 CFTR structure and function.
Topics: Cryoelectron Microscopy; Cystic Fibrosis Transmembrane Conductance Regulator; Phenylalanine; Humans; Sequence Deletion; Protein Conformation; Drug Synergism; Cystic Fibrosis
PubMed: 36264792
DOI: 10.1126/science.ade2216 -
Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing.Science Advances Apr 2021Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor...
Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.
Topics: Animals; CRISPR-Cas Systems; Dystrophin; Exons; Gene Editing; Humans; Induced Pluripotent Stem Cells; Mice; Muscular Dystrophy, Duchenne; Sequence Deletion
PubMed: 33931459
DOI: 10.1126/sciadv.abg4910 -
No Shinkei Geka. Neurological Surgery Sep 2023Meningioma is considered as a single type by the World Health Organization 2021 classification, encompassing 15 subtypes. These classifications are primarily based on...
Meningioma is considered as a single type by the World Health Organization 2021 classification, encompassing 15 subtypes. These classifications are primarily based on histological attributes. However, recent advancements in understanding driver gene mutations and molecular prognostic markers are of particular importance for comprehending the clinical behavior of these tumors. Meningiomas are stratified molecularly, predominantly based on the presence of NF2 mutation. Over 50% are associated with mutations in the NF2 gene and/or monosomy of chromosome 22, whereas the remaining are associated with gene mutations in either of AKT1, SMO, KLF4, TRAF7, or the other less common ones. Importantly, these driver gene mutations are mutually exclusive and significantly associated with tumor location, histological subtype, and grading. The majority of higher-grade meningiomas is associated with multiple chromosomal aberrations, including the loss of 1p, 6q, and 14q, along with NF2 mutations. Homozygous deletion of CDKN2A/CDKN2B genes or TERT promoter mutations serves as a predictor of poor prognosis. Notably, these molecular markers are now incorporated into the criteria for anaplastic meningioma. The amalgamation of molecular insights with clinical and histological parameters, alongside patient prognosis, holds significant utility in the management of patients with meningiomas. It is anticipated to pave the way for treatments founded on molecular-clinical associations.
Topics: Humans; Meningioma; Homozygote; Sequence Deletion; Mutation; Meningeal Neoplasms
PubMed: 37743334
DOI: 10.11477/mf.1436204824 -
Genetics in Medicine : Official Journal... Dec 2019Mediator is a multiprotein complex that allows the transfer of genetic information from DNA binding proteins to the RNA polymerase II during transcription initiation....
PURPOSE
Mediator is a multiprotein complex that allows the transfer of genetic information from DNA binding proteins to the RNA polymerase II during transcription initiation. MED12L is a subunit of the kinase module, which is one of the four subcomplexes of the mediator complex. Other subunits of the kinase module have been already implicated in intellectual disability, namely MED12, MED13L, MED13, and CDK19.
METHODS
We describe an international cohort of seven affected individuals harboring variants involving MED12L identified by array CGH, exome or genome sequencing.
RESULTS
All affected individuals presented with intellectual disability and/or developmental delay, including speech impairment. Other features included autism spectrum disorder, aggressive behavior, corpus callosum abnormality, and mild facial morphological features. Three individuals had a MED12L deletion or duplication. The other four individuals harbored single-nucleotide variants (one nonsense, one frameshift, and two splicing variants). Functional analysis confirmed a moderate and significant alteration of RNA synthesis in two individuals.
CONCLUSION
Overall data suggest that MED12L haploinsufficiency is responsible for intellectual disability and transcriptional defect. Our findings confirm that the integrity of this kinase module is a critical factor for neurological development.
Topics: Adolescent; Autism Spectrum Disorder; Child; Child, Preschool; Developmental Disabilities; Exome; Female; Frameshift Mutation; Humans; Intellectual Disability; Male; Mediator Complex; Mutation; Sequence Deletion; Transcription Factors; Young Adult
PubMed: 31155615
DOI: 10.1038/s41436-019-0557-3 -
Genes Jun 2021Mowat-Wilson Syndrome (MWS) (OMIM # 235730) is a rare disorder due to gene defects (heterozygous mutation or deletion). The gene is a widely expressed regulatory gene,... (Review)
Review
Mowat-Wilson Syndrome (MWS) (OMIM # 235730) is a rare disorder due to gene defects (heterozygous mutation or deletion). The gene is a widely expressed regulatory gene, extremely important for the proper prenatal development. MWS is characterized by a specific facial gestalt and multiple musculoskeletal, cardiac, gastrointestinal, and urogenital anomalies. The nervous system involvement is extensive and constitutes one of the main features in MWS, heavily affecting prognosis and life quality of affected individuals. This review aims to comprehensively organize and discuss the neurological and neurodevelopmental phenotype of MWS. First, we will describe the role of in the formation and development of the nervous system by reviewing the preclinical studies in this regard. regulates the neural crest cell differentiation and migration, as well as in the modulation of GABAergic transmission. This leads to different degrees of structural and functional impairment that have been explored and deepened by various authors over the years. Subsequently, the different neurological aspects of MWS (head and brain malformations, epilepsy, sleep disorders, and enteric and peripheral nervous system involvement, as well as developmental, cognitive, and behavioral features) will be faced one at a time and extensively examined from both a clinical and etiopathogenetic point of view, linking them to the related pathways.
Topics: Abnormalities, Multiple; Embryonic Development; Facies; Genetic Predisposition to Disease; Heterozygote; Hirschsprung Disease; Humans; Intellectual Disability; Microcephaly; Phenotype; Sequence Deletion; Zinc Finger E-box Binding Homeobox 2
PubMed: 34199024
DOI: 10.3390/genes12070982 -
Structure (London, England : 1993) Jun 2023In-frame deletion mutations can result in disease. The impact of these mutations on protein structure and subsequent functional changes remain understudied, partially...
In-frame deletion mutations can result in disease. The impact of these mutations on protein structure and subsequent functional changes remain understudied, partially due to the lack of comprehensive datasets including a structural readout. In addition, the recent breakthrough in structure prediction through deep learning demands an update of computational deletion mutation prediction. In this study, we deleted individually every residue of a small α-helical sterile alpha motif domain and investigated the structural and thermodynamic changes using 2D NMR spectroscopy and differential scanning fluorimetry. Then, we tested computational protocols to model and classify observed deletion mutants. We show a method using AlphaFold2 followed by RosettaRelax performs the best overall. In addition, a metric containing pLDDT values and Rosetta ΔΔG is most reliable in classifying tolerated deletion mutations. We further test this method on other datasets and show they hold for proteins known to harbor disease-causing deletion mutations.
Topics: Proteins; Mutation; Computer Simulation; Sequence Deletion; Magnetic Resonance Spectroscopy; Computational Biology
PubMed: 37119820
DOI: 10.1016/j.str.2023.04.005 -
Microbiology Spectrum Jun 2022The evolutional process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) development remains inconclusive. This study compared the genome sequences of...
The evolutional process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) development remains inconclusive. This study compared the genome sequences of severe acute respiratory syndrome coronavirus (SARS-CoV), bat coronavirus RaTG13, and SARS-CoV-2. In total, the genomes of SARS-CoV-2 and RaTG13 were 77.9% and 77.7% identical to the genome of SARS-CoV, respectively. A total of 3.6% (1,068 bases) of the SARS-CoV-2 genome was derived from insertion and/or deletion (indel) mutations, and 18.6% (5,548 bases) was from point mutations from the genome of SARS-CoV. At least 35 indel sites were confirmed in the genome of SARS-CoV-2, in which 17 were with ≥10 consecutive bases long. Ten of these relatively long indels were located in the spike (S) gene, five in nonstructural protein 3 (Nsp3) gene of open reading frame (ORF) 1a, and one in ORF8 and noncoding region. Seventeen (48.6%) of the 35 indels were based on insertion-and-deletion mutations with exchanged gene sequences of 7-325 consecutive bases. Almost the complete ORF8 gene was replaced by a single 325 consecutive base-long indel. The distribution of these indels was roughly in accordance with the distribution of the rate of point mutation rate around the indels. The genome sequence of SARS-CoV-2 was 96.0% identical to that of RaTG13. There was no long insertion-and-deletion mutation between the genomes of RaTG13 and SARS-CoV-2. The findings of the uneven distribution of multiple indels and the presence of multiple long insertion-and-deletion mutations with exchanged consecutive base sequences in the viral genome may provide insights into SARS-CoV-2 development. The developmental mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains inconclusive. This study compared the base sequence one-by-one between severe acute respiratory syndrome coronavirus (SARS-CoV) or bat coronavirus RaTG13 and SARS-CoV-2. The genomes of SARS-CoV-2 and RaTG13 were 77.9% and 77.7% identical to the genome of SARS-CoV, respectively. Seventeen of the 35 sites with insertion and/or deletion mutations between SARS-CoV-2 and SARS-CoV were based on insertion-and-deletion mutations with the replacement of 7-325 consecutive bases. Most of these long insertion-and-deletion sites were concentrated in the nonstructural protein 3 (Nsp3) gene of open reading frame (ORF) 1a, S1 domain of the spike protein, and ORF8 genes. Such long insertion-and-deletion mutations were not observed between the genomes of RaTG13 and SARS-CoV-2. The presence of multiple long insertion-and-deletion mutations in the genome of SARS-CoV-2 and their uneven distributions may provide further insights into the development of the virus.
Topics: Animals; COVID-19; Chiroptera; Genome, Viral; Phylogeny; SARS-CoV-2; Sequence Deletion
PubMed: 35658573
DOI: 10.1128/spectrum.00716-22 -
Methods in Molecular Biology (Clifton,... 2023Mitochondrial DNA (mtDNA) deletions underpin mitochondrial dysfunction in human tissues in aging and disease. The multicopy nature of the mitochondrial genome means...
Mitochondrial DNA (mtDNA) deletions underpin mitochondrial dysfunction in human tissues in aging and disease. The multicopy nature of the mitochondrial genome means these mtDNA deletions can occur in varying mutation loads. At low levels, these deletions have no impact, but once the proportion of molecules harbouring a deletion exceeds a threshold level, then dysfunction occurs. The location of the breakpoints and the size of the deletion impact upon the mutation threshold required to cause deficiency of an oxidative phosphorylation complex, and this varies for each of the different complexes. Furthermore, mutation load and deletion species can vary between adjacent cells in a tissue, with a mosaic pattern of mitochondrial dysfunction observed. As such, it is often important for understanding human aging and disease to be able to characterise the mutation load, breakpoints and size of deletion(s) from a single human cell. Here, we detail protocols for laser micro-dissection and single cell lysis from tissues, and the subsequent analysis of deletion size, breakpoints and mutation load using long-range PCR, mtDNA sequencing and real-time PCR, respectively.
Topics: Humans; DNA, Mitochondrial; Aging; Mitochondria; Real-Time Polymerase Chain Reaction; Single-Cell Analysis; Sequence Deletion
PubMed: 36807808
DOI: 10.1007/978-1-0716-2922-2_29 -
American Journal of Medical Genetics.... Dec 2019EML1 encodes the protein Echinoderm microtubule-associated protein-like 1 or EMAP-1 that binds to the microtubule complex. Mutations in this gene resulting in complex... (Review)
Review
EML1 encodes the protein Echinoderm microtubule-associated protein-like 1 or EMAP-1 that binds to the microtubule complex. Mutations in this gene resulting in complex brain malformations have only recently been published with limited clinical descriptions. We provide further clinical and imaging details on three previously published families, and describe two novel unrelated individuals with a homozygous partial EML1 deletion and a homozygous missense variant c.760G>A, p.(Val254Met), respectively. From review of the clinical and imaging data of eight individuals from five families with biallelic EML1 variants, a very consistent imaging phenotype emerges. The clinical syndrome is characterized by mainly neurological features including severe developmental delay, drug-resistant seizures and visual impairment. On brain imaging there is megalencephaly with a characteristic ribbon-like subcortical heterotopia combined with partial or complete callosal agenesis and an overlying polymicrogyria-like cortical malformation. Several of its features can be recognized on prenatal imaging especially the abnormaly formed lateral ventricles, hydrocephalus (in half of the cases) and suspicion of a neuronal migration disorder. In conclusion, biallelic EML1 disease-causing variants cause a highly specific pattern of congenital brain malformations, severe developmental delay, seizures and visual impairment.
Topics: Brain; Humans; Malformations of Cortical Development, Group II; Microtubule-Associated Proteins; Mutation, Missense; Sequence Deletion
PubMed: 31710781
DOI: 10.1002/ajmg.c.31751 -
Oral Oncology Sep 2023We aimed to investigate genetic alterations in oral tongue squamous cell carcinoma (OTSCC) based on age and the clinical significance of these alterations in young OTSCC...
OBJECTIVES
We aimed to investigate genetic alterations in oral tongue squamous cell carcinoma (OTSCC) based on age and the clinical significance of these alterations in young OTSCC patients.
MATERIALS AND METHODS
We detected genetic alterations in 44 cases of advanced OTSCC through next-generation sequencing and analyzed and compared patients either younger or older than 45 years. Further analysis was conducted on a validation group of 96 OTSCC patients aged ≤ 45 years to examine the clinical and prognostic associations of TERT promoter (TERTp) mutations.
RESULTS
TP53 mutation was the most common genetic alteration in advanced OTSCC (88.6%), followed by TERTp mutation (59.1%), CDKN2A mutation (31.8%), FAT1 mutation (9.1%), NOTCH1 mutation (9.1%), EGFR amplification (18.2%), and CDKN2A homozygous deletion (4.5%). TERTp mutation was the only genetic alteration significantly enriched in young patients (81.3% in young versus 46.4% in older; P < 0.024). Within the validation group of young patients, TERTp mutation was identified in 30 cases (30/96, 31.3%) and tended to be related to both smoking and alcohol consumption (P = 0.072), higher stage (P = 0.002), more frequent perineural invasion (P = 0.094), and worse overall survival (P = 0.012) than wild type.
CONCLUSION
Our findings suggest that TERTp mutation is more frequent in young patients with advanced OTSCC and is associated with worse clinical outcomes. Therefore, TERTp mutation may serve as a prognostic biomarker for OTSCC in young patients. The findings of this study may help in developing personalized treatment strategies for OTSCC based on age and genetic alterations.
Topics: Humans; Aged; Squamous Cell Carcinoma of Head and Neck; Carcinoma, Squamous Cell; Homozygote; Tongue Neoplasms; Sequence Deletion; Prognosis; Head and Neck Neoplasms
PubMed: 37393663
DOI: 10.1016/j.oraloncology.2023.106466