RESUMO
Five novel HLA-C alleles detected by next-generation sequencing: HLA-C*02:02:73, -C*03:04:106, -C*06:382, -C*07:1114Q and -C*12:408.
Assuntos
Alelos , Antígenos HLA-C , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Antígenos HLA-C/genética , Teste de Histocompatibilidade/métodos , Éxons , Análise de Sequência de DNA/métodosRESUMO
The novel HLA-B*35:593 allele, first described in a potential bone marrow donor from Brazil.
Assuntos
Alelos , Éxons , Humanos , Teste de Histocompatibilidade/métodos , Análise de Sequência de DNA/métodos , Doadores de Tecidos , Brasil , Antígeno HLA-B35/genética , Antígenos HLA-B/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de BasesRESUMO
Expression quantitative trait loci (eQTL) studies typically consider exon expression of genes and discard intronic RNA sequencing reads despite their information on RNA metabolism. Here, we quantify genetic effects on exon and intron levels of genes and their ratio in lymphoblastoid cell lines, revealing thousands of cis-QTLs of each type. While genetic effects are often shared between cis-QTL types, 7814 (47%) are not detected as top cis-QTLs at exon levels. We show that exon levels preferentially capture genetic effects on transcriptional regulation, while exon-intron-ratios better detect those on co- and post-transcriptional processes. Considering all cis-QTL types substantially increases (by 71%) the number of colocalizing variants identified by genome-wide association studies (GWAS). It further allows dissecting the potential gene regulatory processes underlying GWAS associations, suggesting comparable contributions by transcriptional (50%) and co- and post-transcriptional regulation (46%) to complex traits. Overall, integrating intronic RNA sequencing reads in eQTL studies expands our understanding of genetic effects on gene regulatory processes.
Assuntos
Éxons , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Íntrons , Locos de Características Quantitativas , Humanos , Íntrons/genética , Éxons/genética , Transcrição Gênica , Linhagem Celular , Análise de Sequência de RNA/métodos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3mut/mut) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3mut/mut mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness.
Assuntos
Sistemas CRISPR-Cas , Distrofias Hereditárias da Córnea , Família 4 do Citocromo P450 , Edição de Genes , Terapia Genética , Células-Tronco Pluripotentes Induzidas , Doenças Retinianas , Humanos , Edição de Genes/métodos , Animais , Células HEK293 , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/terapia , Distrofias Hereditárias da Córnea/patologia , Distrofias Hereditárias da Córnea/metabolismo , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Terapia Genética/métodos , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Mutação , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Vetores Genéticos/genética , Íntrons/genética , Éxons/genéticaRESUMO
The novel HLA-A*02:829 allele is likely generated from the recombination of both HLA-A*02:05:01:01 and HLA-A*32:01:01:01 alleles.
Assuntos
Alelos , Sequência de Bases , Éxons , Antígeno HLA-A2 , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Análise de Sequência de DNA/métodos , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Alinhamento de Sequência , Recombinação Genética , CódonRESUMO
MAPK activating death domain (MADD) is a multifunctional protein regulating small GTPases RAB3 and RAB27, MAPK signaling, and cell survival. Polymorphisms in the MADD locus are associated with glycemic traits, but patients with biallelic variants in MADD manifest a complex syndrome affecting nervous, endocrine, exocrine, and hematological systems. We identified a homozygous splice site variant in MADD in 2 siblings with developmental delay, diabetes, congenital hypogonadotropic hypogonadism, and growth hormone deficiency. This variant led to skipping of exon 30 and in-frame deletion of 36 amino acids. To elucidate how this mutation causes pleiotropic endocrine phenotypes, we generated relevant cellular models with deletion of MADD exon 30 (dex30). We observed reduced numbers of ß cells, decreased insulin content, and increased proinsulin-to-insulin ratio in dex30 human embryonic stem cell-derived pancreatic islets. Concordantly, dex30 led to decreased insulin expression in human ß cell line EndoC-ßH1. Furthermore, dex30 resulted in decreased luteinizing hormone expression in mouse pituitary gonadotrope cell line LßT2 but did not affect ontogeny of stem cell-derived GnRH neurons. Protein-protein interactions of wild-type and dex30 MADD revealed changes affecting multiple signaling pathways, while the GDP/GTP exchange activity of dex30 MADD remained intact. Our results suggest MADD-specific processes regulate hormone expression in pancreatic ß cells and pituitary gonadotropes.
Assuntos
Células Secretoras de Insulina , Células Secretoras de Insulina/metabolismo , Humanos , Animais , Camundongos , Masculino , Gonadotrofos/metabolismo , Feminino , Sítios de Splice de RNA/genética , Linhagem Celular , Insulina/metabolismo , Irmãos , Éxons/genética , Proteínas rab3 de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/genética , Hipogonadismo/genética , Hipogonadismo/metabolismo , Hipogonadismo/patologiaRESUMO
Recently, targeted therapy and immunotherapy have emerged as effective treatment options for non-small cell lung cancer (NSCLC). This progress has been facilitated by the rapid development of diagnostic and therapeutic technologies and the continuous research and development of new drugs, leading to a new era in precision medicine for NSCLC. This is a breakthrough for patients with common mutations in the human epidermal growth factor receptor (EGFR) gene in NSCLC. Consequently, the use of targeted drugs has significantly improved survival. Nevertheless, certain rare genetic mutations are referred to as EGFR exon 20 insertion (ex20ins) mutations, which differ in structure from conventional EGFR gene mutations, namely, exon 19 deletion mutations (19-Del) and exon 21 point mutations. Owing to their distinct structural characteristics, patients harboring these EGFR ex20ins mutations are unresponsive to traditional tyrosine kinase inhibitor (TKI) therapy. This particular group of patients did not fall within the scope of their applicability. However, the activating A763_Y764insFQEA mutation elicits a more pronounced response than mutations in the near and far regions of the C-helix immediately following it and should, therefore, be treated differently. Currently, there is a lack of effective treatments for EGFR ex20ins mutations NSCLC. The efficacy of chemotherapy has been relatively favorable, whereas the effectiveness of immunotherapy remains ambiguous owing to inadequate clinical data. In addition, the efficacy of the first- and second-generation targeted drugs remains limited. However, third-generation and novel targeted drugs have proven to be effective. Although novel EGFR-TKIs are expected to treat EGFR ex20ins mutations in patients with NSCLC, they face many challenges. The main focus of this review is on emerging therapies that target NSCLC with EGFR ex20ins and highlight major ongoing clinical trials while also providing an overview of the associated challenges and research advancements in this area.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Receptores ErbB , Éxons , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/terapia , Receptores ErbB/genética , Receptores ErbB/antagonistas & inibidores , Éxons/genética , Inibidores de Proteínas Quinases/uso terapêutico , Imunoterapia/métodos , Mutagênese Insercional , Terapia de Alvo Molecular , Mutação , AnimaisRESUMO
Breast cancer (BC) is a leading cause of cancer deaths in Libyan women. BRCA1 variants differ globally due to the diversity of genetic makeup and populations history. Their distribution, prevalence, and significance in Libyans remain largely unexplored. This study investigated the characteristics and distribution of BRCA1 variants in exons 5, 11, and 20 in Libyan families with BC. Thirty-six BC patients at ≤ 45 years, between 46-50 years and with a family history of breast, ovarian, pancreatic or prostate cancer in close relatives, or with triple-negative BC, were selected from 33 unrelated families during 2018-2020 at the National Cancer Institute, Sabratha, Libya. From these 33 families, 20 women (18 BC patients and two unaffected) were screened for BRCA1 exons 5, 11 and 20 using Sanger sequencing. All families completed an epidemiology and family history questionnaire. Twenty-seven variants (26 in exon 11 and 1 in exon 20, minor allele frequency of < 0.01) were detected in 10 of 18 unrelated families (55.6%.) Among the 27 variants, 26 (96%) were heterozygous. A frameshift pathogenic variant, c.2643del, and one novel variant c.1366A>G were identified. Furthermore, seven variants with unknown clinical significance were detected: c.1158T>A, c.1346C>G, c.1174C>G, c.3630 G>T, c.3599A>T, and c.3400 G>C in exon 11, and c.5244T>A in exon 20. Six variants with conflicting pathogenicity interpretations, c. 3460T>A, c. 3572 G>A, c. 3700 G>C, c. 1246C>G, c. 1344C>G, and c. 1054 G>A, were also identified. Twelve benign/likely benign variants were identified. Rare BRCA1 variants that have not been reported in North Africa were found in Libyan patients. These findings provide preliminary insights into the BRCA1 variants that could contribute to hereditary BC risk in Libyans. Further functional, computational, and population analyses are essential to determine their significance and potential impact on BC risk, which could ultimately lead to more personalized management strategies.
Assuntos
Proteína BRCA1 , Neoplasias da Mama , Éxons , Mutação em Linhagem Germinativa , Humanos , Líbia/epidemiologia , Feminino , Pessoa de Meia-Idade , Neoplasias da Mama/genética , Neoplasias da Mama/epidemiologia , Proteína BRCA1/genética , Adulto , Predisposição Genética para Doença , Frequência do GeneRESUMO
HLA-C*03:620 differs from the HLA-C*03:04:01:02 allele by one nucleotide substitution in the exon 3.
Assuntos
Alelos , Povo Asiático , Sequência de Bases , Éxons , Antígenos HLA-C , Teste de Histocompatibilidade , Humanos , Antígenos HLA-C/genética , Povo Asiático/genética , Análise de Sequência de DNA/métodos , Códon , Alinhamento de Sequência , Polimorfismo de Nucleotídeo Único , População do Leste AsiáticoRESUMO
HLA-DPB1*05:01:20 differs from HLA-DPB1*05:01:01:01 by one nucleotide in exon 3.
Assuntos
Alelos , Povo Asiático , Sequência de Bases , Éxons , Cadeias beta de HLA-DP , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Cadeias beta de HLA-DP/genética , Povo Asiático/genética , Análise de Sequência de DNA/métodos , Doadores de Tecidos , Alinhamento de Sequência , Códon , População do Leste AsiáticoRESUMO
HLA-B*15:659 differs from HLA-B*15:02:01:01 by one nucleotide in exon 2.
Assuntos
Alelos , Povo Asiático , Sequência de Bases , Éxons , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Povo Asiático/genética , Análise de Sequência de DNA/métodos , Antígeno HLA-B15/genética , Antígeno HLA-B15/imunologia , Alinhamento de Sequência , Códon , Doadores de Tecidos , População do Leste AsiáticoRESUMO
Genomic full-length sequence of HLA-B*37:46 was identified by a group-specific sequencing approach in a Chinese individual.
Assuntos
Alelos , Povo Asiático , Antígenos HLA-B , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Antígenos HLA-B/genética , Análise de Sequência de DNA/métodos , Teste de Histocompatibilidade/métodos , Povo Asiático/genética , Éxons , Sequência de BasesRESUMO
The novel HLA-A*02:1140 allele, first described in a potential bone marrow donor from Brazil.
Assuntos
Alelos , Antígeno HLA-A2 , Humanos , Brasil , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Teste de Histocompatibilidade , Éxons , Doadores de Tecidos , Sequência de Bases , Análise de Sequência de DNA/métodos , Alinhamento de SequênciaRESUMO
Eukaryotic DNA codes not only for proteins but contains a wealth of information required for accurate splicing of messenger RNA precursors and inclusion of constitutively or alternatively spliced exons in mature transcripts. This "auxiliary" splicing code has been characterized as exonic splicing enhancers and silencers (ESE and ESS). The exact interplay between protein and splicing codes is, however, poorly understood. Here, we show that exons encoding copper-coordinating amino acids in human cuproproteins lack ESEs and/or have an excess of ESSs, yet RNA sequencing and expressed sequence tags data show that they are more efficiently included in mature transcripts by the splicing machinery than average exons. Their largely constitutive inclusion in messenger RNA is facilitated by stronger splice sites, including polypyrimidine tracts, consistent with an important role of the surrounding intron architecture in ensuring high expression of metal-binding residues during evolution. ESE/ESS profiles of codons and entire exons that code for copper-coordinating residues were very similar to those encoding residues that coordinate zinc but markedly different from those that coordinate calcium. Together, these results reveal how the traditional and auxiliary splicing motifs responded to constraints of metal coordination in proteins.
Assuntos
Cobre , Éxons , Splicing de RNA , Humanos , Éxons/genética , Cobre/metabolismo , Processamento Alternativo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismoRESUMO
HLA-A*01:454 and HLA-A*31:229, two novel HLA-A alleles detected during routine typing by next-generation sequencing.
Assuntos
Alelos , Éxons , Antígenos HLA-A , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Humanos , Antígenos HLA-A/genética , Análise de Sequência de DNA/métodos , Antígeno HLA-A1/genética , Sequência de BasesRESUMO
Objective: To analyze the distribution characteristics of UGT1A1 mutant genes (including enhancers, promoters, and exons 1-5) and further explore the correlation between UGT1A1 genotype and clinical phenotypes in patients with inherited hyperunconjugated bilirubinemia. Methods: Patients diagnosed with hereditary hyperunconjugated bilirubinemia at Nanjing Second Hospital from June 2015 to December 2022 were retrospectively analyzed. The UGT1A1 gene was examined using Sanger sequencing in all patients. Complete blood count, liver function, and abdominal imaging examinations were performed. Comparison of categorical variable data using χ(2) testor Fisher percision tests. Comparison of continaous veriable data with normal distribution using t-test. Results: 112 cases (male:female ratio 81:31, aged 9-70 years) had inherited hyperunconjugated bilirubinemia, with a total of 14 mutation sites identified, of which seven were confirmed mutations, and the frequency ranged from high to low: (TA)n accounted for 50%, c.211G>A (p.G71R) accounted for 49.10%, 1456T>G (p.Y486D) accounted for 16.96%, c.686C>A (p.R229W) accounted for 12.5%, 1091C>T (p.P364L) accounted for 8.04%, and c- 3279T>G accounted for 0.982%. Simultaneously, all patients had one to four mutations, of which only one mutation was the most common (55.36%), followed by two mutations (37.5%), and rare three and four mutations (5.36% and 1.78%). There was no statistical significance in total bilirubin (TBil) levels among the four groups (F=0.652, P=0.583). One mutation was most common in (TA)n and c.211G>A (p.G71R), among which TA6/TA7 (n=10) and TA7/TA7 (n=14) mutations were statistically significant in TBil (t=2.143, P=0.043). The c.211G>A (p.G71R) heterozygous (n=9) and isolated (n=15) mutation had no statistical significance in TBil (t=0.382, P=0.706). The GS group accounted for 75%, the intermediate group accounted for 16.9%, and the CNS-â ¡ group accounted for 8%. TBil was statistically significant among the three groups (F=270.992, P<0.001). There was no statistically significant difference (χ(2)=3.317, P=0.19) between mutation 1 (44 cases, 14 cases, and 4 cases, respectively) and mutations ≥ 2 (40 cases, 5 cases, and 5 cases, respectively) in the GS group, intermediate group, and CNS-II group. Conclusion: The number of UGT1A1 gene mutation sites may have no synergistic effect on TBil levels in patients with inherited hyperunconjugated bilirubinemia. TA7/TA7 mutations are not uncommon, and TBil levels are relatively high.
Assuntos
Genótipo , Glucuronosiltransferase , Mutação , Fenótipo , Humanos , Glucuronosiltransferase/genética , Estudos Retrospectivos , Hiperbilirrubinemia Hereditária/genética , Bilirrubina/sangue , Masculino , Feminino , Éxons , AdultoRESUMO
HLA-DQA1*02:32 differs from DQA1*02:01:01 by one nucleotide substitution in codon 210 in exon 4.
Assuntos
Alelos , Éxons , Cadeias alfa de HLA-DQ , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Humanos , Cadeias alfa de HLA-DQ/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade/métodos , Sequência de Bases , Códon , Análise de Sequência de DNA/métodosRESUMO
The novel HLA-C*15:274 allele, first described in a potential bone marrow donor from Brazil.
Assuntos
Alelos , Éxons , Antígenos HLA-C , Teste de Histocompatibilidade , Humanos , Antígenos HLA-C/genética , Teste de Histocompatibilidade/métodos , Análise de Sequência de DNA/métodos , Doadores de Tecidos , Brasil , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de BasesRESUMO
The novel HLA-DPA1*02:07:04 allele was detected during the HLA typing for kidney transplantation.
Assuntos
Alelos , Cadeias alfa de HLA-DP , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Transplante de Rim , Humanos , Cadeias alfa de HLA-DP/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade/métodos , ÉxonsRESUMO
Immunotherapy with chimeric antigen receptor T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify cancer specific exon targets, here we analyze 1532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We find 2933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n = 148) or the alternatively spliced isoform (n = 9) level. Expression of selected alternatively spliced targets, including the EDB domain of fibronectin 1, and gene targets, such as COL11A1, are validated in pediatric patient derived xenograft tumors. We generate T cells expressing chimeric antigen receptors specific for the EDB domain or COL11A1 and demonstrate that these have antitumor activity. The full target list, explorable via an interactive web portal ( https://cseminer.stjude.org/ ), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.