RESUMEN
Oprm1, the gene encoding the µ-opioid receptor, has multiple reported transcripts, with a variable 3' region and many alternative sequences encoding the C-terminus of the protein. The functional implications of this variability remain mostly unexplored, though a recurring notion is that it could be exploited by developing selective ligands with improved clinical profiles. Here, we comprehensively examined Oprm1 transcriptional variants in the murine central nervous system, using long-read RNAseq as well as spatial and single-cell transcriptomics. The results were validated with RNAscope in situ hybridization. We found a mismatch between transcripts annotated in the mouse genome (GRCm38/mm10) and the RNA-seq results. Sequencing data indicated that the primary Oprm1 transcript has a 3' terminus located on chr10:6,860,027, which is â¼ 9.5 kilobases downstream of the longest annotated exon 4 end. Long-read sequencing confirmed that the final Oprm1 exon included a 10.2 kilobase long 3' untranslated region, and the presence of the long variant was unambiguously confirmed using RNAscope in situ hybridization in the thalamus, striatum, cortex and spinal cord. Conversely, expression of the Oprm1 reference transcript or alternative transcripts of the Oprm1 gene was absent or close to the detection limit. Thus, the primary transcript of the Oprm1 mouse gene is a variant with a long 3' untranslated region, which is homologous to the human OPRM1 primary transcript and encodes the same conserved C-terminal amino acid sequence.
Asunto(s)
Prosencéfalo , Receptores Opioides mu , Médula Espinal , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Animales , Ratones , Médula Espinal/metabolismo , Prosencéfalo/metabolismo , Masculino , Ratones Endogámicos C57BL , Exones , Regiones no Traducidas 3'Asunto(s)
Dermatitis Atópica , Exones , Proteínas Filagrina , Proteínas de Filamentos Intermediarios , Humanos , Dermatitis Atópica/genética , Dermatitis Atópica/diagnóstico , Proteínas de Filamentos Intermediarios/genética , Exones/genética , Masculino , Femenino , Mutación con Pérdida de Función , Adulto , Índice de Severidad de la EnfermedadRESUMEN
The novel KIR2DL1*00308 allele differs from the closest allele KIR2DL1*00302 by a single sense mutation.
Asunto(s)
Alelos , Pueblo Asiatico , Exones , Receptores KIR2DL1 , Humanos , Pueblo Asiatico/genética , Receptores KIR2DL1/genética , Secuencia de Bases , Análisis de Secuencia de ADN , China , Alineación de Secuencia , Prueba de Histocompatibilidad , Pueblos del Este de AsiaRESUMEN
The HLA-A*23:140 allele differs from HLA-A*23:01:01 by one nucleotide substitution (G > A), position 1968 in exon 5.
Asunto(s)
Alelos , Exones , Antígenos HLA-A , Donantes de Tejidos , Humanos , Brasil , Antígenos HLA-A/genética , Prueba de Histocompatibilidad , Secuencia de Bases , Análisis de Secuencia de ADN/métodos , Polimorfismo de Nucleótido Simple , Médula Ósea , Trasplante de Médula ÓseaRESUMEN
HLA-DPA1*01:12:03 differs from HLA-DPA1*01:12:01 by one nucleotide substitution in codon 204 in exon 4.
Asunto(s)
Alelos , Secuencia de Bases , Exones , Cadenas alfa de HLA-DP , Prueba de Histocompatibilidad , Análisis de Secuencia de ADN , Humanos , Cadenas alfa de HLA-DP/genética , Cadenas alfa de HLA-DP/inmunología , Prueba de Histocompatibilidad/métodos , Análisis de Secuencia de ADN/métodos , Codón , Alineación de SecuenciaRESUMEN
HLA-C*07:02:81 differs from HLA-C*07:02:01:01 by one nucleotide substitution at position 465 (CâA) in exon 3.
Asunto(s)
Alelos , Secuencia de Bases , Exones , Antígenos HLA-C , Prueba de Histocompatibilidad , Humanos , Antígenos HLA-C/genética , Análisis de Secuencia de ADN , Mutación Silenciosa , Alineación de Secuencia , CodónRESUMEN
Two novel Class I HLA-B alleles HLA-B*13:194 and HLA-B*15:694 are described.
Asunto(s)
Alelos , Exones , Humanos , Lituania , Secuencia de Bases , Prueba de Histocompatibilidad , Análisis de Secuencia de ADN , Antígeno HLA-B13/genética , Antígeno HLA-B13/inmunología , Antígeno HLA-B15/genética , Antígeno HLA-B15/inmunología , Antígenos HLA-B/genética , Alineación de Secuencia , CodónRESUMEN
Identification of seven new HLA alleles by next-generation sequencing.
Asunto(s)
Alelos , Antígenos HLA , Secuenciación de Nucleótidos de Alto Rendimiento , Prueba de Histocompatibilidad , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Antígenos HLA/genética , Prueba de Histocompatibilidad/métodos , Exones , Análisis de Secuencia de ADN/métodos , Secuencia de BasesRESUMEN
Prostate cancer (PCa) is the most common cancer diagnosed in men worldwide and was the second leading cause of cancer-related deaths in US males in 2022. Prostate cancer also represents the second highest cancer mortality disparity between non-Hispanic blacks and whites. However, there is a relatively small number of prostate normal and cancer cell lines compared to other cancers. To identify the molecular basis of PCa progression, it is important to have prostate epithelial cell (PrEC) lines as karyotypically normal as possible. Our lab recently developed a novel methodology for the rapid and efficient immortalization of normal human PrEC that combines simultaneous CRISPR-directed inactivation of CDKN2A exon 2 (which directs expression of p16INK4A and p14ARF) and ectopic expression of an hTERT transgene. To optimize this methodology to generate immortalized lines with minimal genetic alterations, we sought to target exon 1α of the CDKN2A locus so that p16INK4A expression is ablated while the exons encoding p14ARF remains unaltered. Here we describe the establishment of two cell lines: one with the above-mentioned p16INK4A only loss, and a second line targeting both products in the CDKN2A locus. We characterize the potential lineage origin of these new cell lines along with our previously obtained clones, revealing distinct gene expression signatures. Based on the analyses of protein markers and RNA expression signatures, these cell lines are most closely related to a subpopulation of basal prostatic cells. Given the simplicity of this one-step methodology and the fact that it uses only the minimal genetic alterations necessary for immortalization, it should also be suitable for the establishment of cell lines from primary prostate tumor samples, an urgent need given the limited number of available prostate cancer cell lines.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina , Células Epiteliales , Próstata , Neoplasias de la Próstata , Telomerasa , Humanos , Masculino , Telomerasa/genética , Telomerasa/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Células Epiteliales/metabolismo , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Exones/genética , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Línea CelularRESUMEN
HLA-DQA1*01:02:24 differs from HLA-DQA1*01:02:01:03 by one nucleotide substitution in codon 167 in exon 3.
Asunto(s)
Alelos , Secuencia de Bases , Exones , Cadenas alfa de HLA-DQ , Prueba de Histocompatibilidad , Análisis de Secuencia de ADN , Humanos , Cadenas alfa de HLA-DQ/genética , Análisis de Secuencia de ADN/métodos , Codón , Alineación de SecuenciaRESUMEN
HLA-B*49:01:01:22 differs from HLA-B*49:01:01:01 by a single nucleotide C->G change in intron 5 at gDNA 2451.
Asunto(s)
Alelos , Secuenciación de Nucleótidos de Alto Rendimiento , Prueba de Histocompatibilidad , Intrones , Trasplante de Riñón , Donantes de Tejidos , Humanos , Prueba de Histocompatibilidad/métodos , India , Exones , Secuencia de Bases , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Antígeno HLA-B39/genéticaRESUMEN
OBJECTIVE: To explore the genetic basis for a child with pachygyria. METHODS: A proband who had visited Qinzhou Maternal and Child Health Care Hospital for pachygyria and mental retardation in June 2020 was selected as the study subject. Clinical data was collected. The child was subjected to whole exome sequencing (WES), and candidate variant was verified by Sanger sequencing. RESULTS: The proband, a 4-year-and-6-month-old female, was clinically diagnosed with megagyrus deformity. WES revealed that she has harbored compound heterozygous variants of the ADGRG1 gene, namely c.781G>T (p.E261*) in exon 6 and c.1369A>C (p.S457R) in exon 11, which were verified by Sanger sequencing to be derived from her mother and father, respectively. Her younger sister was also heterozygous for the c.1369A>C (p.S457R) variant. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were rated as likely pathogenic (PVS1+PM2_ Supporting; PM1+PM2_Supporting+PM3+PP3). CONCLUSION: The c.781G>T (p.E261*) and c.1369A>C (p.S457R) compound heterozygous variants of the ADGRG1 gene probably underlay the pachygyria malformation in this child.
Asunto(s)
Secuenciación del Exoma , Humanos , Femenino , Preescolar , Heterocigoto , Mutación , Lisencefalia/genética , Pruebas Genéticas , Exones , Discapacidad Intelectual/genética , LinajeRESUMEN
The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications.
Asunto(s)
Adenosina , Citosina , Edición Génica , Distrofia Muscular de Duchenne , Citosina/metabolismo , Animales , Edición Génica/métodos , Adenosina/metabolismo , Ratones , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Ingeniería de Proteínas , Distrofina/genética , Distrofina/metabolismo , Adenosina Desaminasa/metabolismo , Adenosina Desaminasa/genética , Modelos Animales de Enfermedad , Exones/genética , Células HEK293 , Sistemas CRISPR-Cas , Proteínas de Escherichia coliRESUMEN
Over 70% of leiomyoma (LM) harbor MED12 mutations, primarily in exon 2 at c.130-131 (GG). Myometrial cells are the cell origin of leiomyoma, but the MED12 mutation status in non-neoplastic myometrial cells is unknown. In this study, we investigated the mutation burden of MED12 in myometrium. As traditional Sanger or even NGS sequencing may not be able to detect MED12 mutations that are lower than 0.1% in the testing sample, we used duplex deep sequencing analysis (DDS) to overcome this limitation. Tumor-free myometria (confirmed by pathology evaluation) were dissected, and genomic DNA from MED12 exon 2 (test) and TP53 exon 5 (control) were captured by customer-designed probe sets, followed by DDS. Notably, DDS demonstrated that myometrial cells harbored a high frequency of mutations in MED12 exon 2 and predominantly in code c.130-131. In contrast, the baseline mutations in other coding sequences of MED12 exon 2 as well as in the TP53 mutation hotspot, c.477-488 were comparably low in myometrial cells. This is the first report demonstrating a non-random accumulation of MED12 mutations at c.130-131 sites in non-neoplastic myometrial cells which provide molecular evidence of early somatic mutation events in myometrial cells. This early mutation may contribute to the cell origin for uterine LM development in women of reproductive age.
Asunto(s)
Complejo Mediador , Mutación , Miometrio , Humanos , Femenino , Miometrio/metabolismo , Miometrio/patología , Complejo Mediador/genética , Complejo Mediador/metabolismo , Mutación/genética , Exones/genética , Leiomioma/genética , Leiomioma/patología , Persona de Mediana Edad , Adulto , Neoplasias Uterinas/genética , Neoplasias Uterinas/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
HLA-DPB1*04:02:24 differs from HLA-DPB1*04:02:01:01 by a single synonmous nucleotide substitution at position 639 G>A.
Asunto(s)
Alelos , Cadenas beta de HLA-DP , Secuenciación de Nucleótidos de Alto Rendimiento , Prueba de Histocompatibilidad , Humanos , Cadenas beta de HLA-DP/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Prueba de Histocompatibilidad/métodos , Exones , Secuencia de Bases , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , CodónRESUMEN
One nucleotide deletion in codon 15 of HLA-B*40:01:02:01 results in a novel null allele, HLA-B*40:510N.
Asunto(s)
Alelos , Exones , Prueba de Histocompatibilidad , Eliminación de Secuencia , Humanos , Secuencia de Bases , Análisis de Secuencia de ADN/métodos , Antígeno HLA-B40/genética , Codón , Antígenos HLA-B/genéticaRESUMEN
HLA-C*08:291, a novel HLA class I allele detected by next-generation sequencing.
Asunto(s)
Alelos , Exones , Antígenos HLA-C , Secuenciación de Nucleótidos de Alto Rendimiento , Prueba de Histocompatibilidad , Humanos , Antígenos HLA-C/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Prueba de Histocompatibilidad/métodos , Secuencia de Bases , Codón , Análisis de Secuencia de ADN/métodos , Alineación de SecuenciaRESUMEN
HLA-DQA1*05:115 shows an alanine at position 59 not described previously.
Asunto(s)
Alanina , Alelos , Cadenas alfa de HLA-DQ , Humanos , Cadenas alfa de HLA-DQ/genética , Alanina/genética , Exones , Prueba de Histocompatibilidad , Sustitución de Aminoácidos , Dominios ProteicosRESUMEN
BACKGROUND: Deletion or duplication in the DMD gene is one of the most common causes of Duchenne and Becker muscular dystrophy (DMD/BMD). However, the pathogenicity of complex rearrangements involving DMD, especially segmental duplications with unknown breakpoints, is not well understood. This study aimed to evaluate the structure, pattern, and potential impact of rearrangements involving DMD duplication. METHODS: Two families with DMD segmental duplications exhibiting phenotypical differences were recruited. Optical genome mapping (OGM) was used to explore the cryptic pattern of the rearrangements. Breakpoints were validated using long-range polymerase chain reaction combined with next-generation sequencing and Sanger sequencing. RESULTS: A multi-copy duplication involving exons 64-79 of DMD was identified in Family A without obvious clinical symptoms. Family B exhibited typical DMD neuromuscular manifestations and presented a duplication involving exons 10-13 of DMD. The rearrangement in Family A involved complex in-cis tandem repeats shown by OGM but retained a complete copy (reading frame) of DMD inferred from breakpoint validation. A reversed insertion with a segmental repeat was identified in Family B by OGM, which was predicted to disrupt the normal structure and reading frame of DMD after confirming the breakpoints. CONCLUSIONS: Validating breakpoint and rearrangement pattern is crucial for the functional annotation and pathogenic classification of genomic structural variations. OGM provides valuable insights into etiological analysis of DMD/BMD and enhances our understanding for cryptic effects of complex rearrangements.
Asunto(s)
Distrofina , Exones , Distrofia Muscular de Duchenne , Linaje , Fenotipo , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Distrofina/genética , Masculino , Exones/genética , Femenino , Mapeo Cromosómico , Reordenamiento Génico/genética , Niño , Duplicaciones Segmentarias en el Genoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Duplicación de Gen/genética , AdolescenteRESUMEN
HLA-DPB1*21:01:02 differs from HLA-DPB1*21:01:01 by one single nucleotide substitution at position 435 C>T in exon 3.