RESUMEN
When a de novo balanced reciprocal translocation is identified in the patient, the cause of phenotype of the patient can be explained by detecting the breakpoints of the genes. Here, we report a 3-year-old patient with developmental delay, autism spectrum disorder, and distinctive facial features who had an apparently balanced translocation between chromosome 3q26 and chromosome 7q36. Nanopore long-read sequencing revealed that balanced translocation disrupted the KMT2C gene, the haploinsufficiency of which leads to Kleefstra syndrome 2 characterized by delayed psychomotor development, variable intellectual disability and mild dysmorphism. Nanopore long-read sequencing was shown to be useful in elucidating the exact genetic etiology of patients with nonspecific clinical findings.
Asunto(s)
Trastorno del Espectro Autista , Discapacidad Intelectual , Humanos , Trastorno del Espectro Autista/diagnóstico , Trastorno del Espectro Autista/genética , Cromosomas , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Fenotipo , Translocación Genética , PreescolarRESUMEN
Abnormal expansion or shortening of tandem repeats can cause a variety of genetic diseases. The use of long DNA reads has facilitated the analysis of disease-causing repeats in the human genome. Long read sequencers enable us to directly analyze repeat length and sequence content by covering whole repeats; they are therefore considered suitable for the analysis of long tandem repeats. Here, we describe an expanded repeat analysis using target sequencing data produced by the Oxford Nanopore Technologies (hereafter referred to as ONT) nanopore sequencer.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Nanoporos , Humanos , Secuencias Repetidas en Tándem/genética , Análisis de Secuencia de ADN , ADN/genéticaRESUMEN
When a de novo balanced reciprocal translocation is identified in patients with multiple congenital abnormalities, attempts are often made to infer the relationship between the phenotype of the patient and genes in the proximity of the breakpoint. Here, we report a patient with intellectual disability, atrial septal defect, syndactyly, and cleft lip and palate who had an "apparently balanced" de novo reciprocal translocation t(4:18)(q31;q11.2) as well as a 7-Mb cryptic deletion spanning the HOXD cluster on chromosome 2q31 that was unrelated to the reciprocal translocation. Further analysis using a nanopore long-read sequencer showed complex rearrangements on both derivative chromosomes 4 and 18 and the deleted chromosome 2. First, the TLL1 locus, which is associated with atrial septal defect, was disrupted by the rearrangement involving chromosome 4. Second, the deleted interval at 2q31 included the entire HOXD cluster, the deletion of which is known to cause toe syndactyly, and the DLX1 and DLX2 loci, which are responsible for cleft lip and palate. Among the haplo-sensitive genes within the deleted interval on 2q31, only the RAPGEF4 gene is known to be associated with an autistic phenotype. Hence, most of the clinical features of the patient could be ascribed to specific genomic rearrangements. We have shown the effectiveness of long-read sequencing in defining, in detail, the likely effects of an apparently balanced translocation and cryptic deletion. The results of the present analysis suggest the possibility of phenotypic prediction through a detailed analysis of structural abnormalities, including balanced translocations and deletions.
Asunto(s)
Labio Leporino , Fisura del Paladar , Defectos del Tabique Interatrial , Sindactilia , Labio Leporino/genética , Fisura del Paladar/genética , Factores de Intercambio de Guanina Nucleótido/genética , Defectos del Tabique Interatrial/diagnóstico , Defectos del Tabique Interatrial/genética , Humanos , Sindactilia/genética , Metaloproteinasas Similares a Tolloid/genética , Translocación GenéticaRESUMEN
BACKGROUND: It has been suggested that the local microbiota in the reproductive organs is relevant to women's health and may also affect pregnancy outcomes. Analysis of partial 16S ribosomal RNA (rRNA) gene sequences generated by short-read sequencers has been used to identify vaginal and endometrial microbiota, but it requires a long time to obtain the results, making it unsuitable for rapid bacterial identification from a small specimen amount in a clinical context. METHODS: We developed a simple workflow using the nanopore sequencer MinION that allows high-resolution and rapid differentiation of vaginal microbiota. Vaginal samples collected from 18 participants were subjected to DNA extraction and full-length 16S rRNA gene sequencing with MinION. RESULTS: The principal coordinate analysis showed no differences in the bacterial compositions regardless of the sample collection method. The analysis of vaginal microbiota could be completed with a total analysis time of approximately four hours, allowing same-day results. Taxonomic profiling by MinION sequencing revealed relatively low diversity of the vaginal bacterial community, identifying the prevailing Lactobacillus species and several causative agents of bacterial vaginosis. CONCLUSIONS: Full-length 16S rRNA gene sequencing analysis with MinION provides a rapid means for identifying vaginal bacteria with higher resolution. Species-level profiling of human vaginal microbiota by MinION sequencing can allow the analysis of associations with conditions such as genital infections, endometritis, and threatened miscarriage.
Asunto(s)
Microbiota , Secuenciación de Nanoporos , Bacterias/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Microbiota/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN/métodosRESUMEN
BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Genes MHC Clase I/genética , Escape del Tumor/genética , Escape del Tumor/inmunología , Microglobulina beta-2/genética , Alelos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Metilación de ADN , Epigénesis Genética , Expresión Génica , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Humanos , Inmunogenética , Linfocitos Infiltrantes de Tumor , Inestabilidad de Microsatélites , Complejo de la Endopetidasa Proteasomal/genética , Factores de Transcripción del Factor Regulador X/genética , Tasa de Supervivencia , Microglobulina beta-2/metabolismoRESUMEN
Transgene insertion patterns are critical for the analysis of transgenic animals because the influence of transgenes may change depending on the insertion pattern (such as copy numbers and orientations of concatenations) and the insertion position in the genome. We previously reported a genomic walking strategy to locate transgenes in the genomes of transgenic mice (Exp. Anim. 53: 103-111, 2004) and to analyze transgene insertion patterns (Exp. Anim. 55: 65-69, 2006). With such strategies, however, we could not determine the copy number of transgenes or global genome modification induced by transgene insertion due to read-length limitation. In this study, we used a long-read sequencer (MinION, Oxford Nanopore Technologies) to overcome this limitation. We obtained 922,210 reads using MinION with genomic DNA from a transgenic mouse strain (4C30, Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 87: 550-562, 2011). Among the reads, we found one 21,457-bp read containing the transgene using a local BLAST search. Nucleotide dot plot analysis revealed that the transgene was inserted in the genome as a tandem concatemer with an almost entire construct (15-3,508 of 3,508 bp) and a partial fragment (4-660, 657 bp). Ensembl's BLAST search against the C57BL/6N genome revealed a 9,388-bp deletion at the insertion position in the intron of the Sgcd gene, confirming that mutations such as a large genomic deletion could occur at the time of transgene insertion. Thus, long-read sequencers are useful tools for the analysis of transgene insertion patterns.
Asunto(s)
Mutagénesis Insercional , Análisis de Secuencia de ADN/métodos , Transgenes/genética , Animales , Genoma/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Sarcoglicanos/genéticaRESUMEN
CRISPR-Cas9 technology has been used in various studies; however, it has also been found to introduce unexpected structural alternations. In this study, we used nanopore sequencing to characterize an unexpected structural alteration of mirror-image duplicated genes in a mouse line, in which we aimed to delete a part of the duplicated genes using genome editing. We removed low-molecular-weight DNA fragments and increased the input, which led to improved sequence performance. With 14.9 Gb input for whole-genome analysis, we detected a complex structural alteration involving inversion and deletion, which appears to be difficult to characterize with short-read sequencers. Therefore, our study clearly showed the utility of nanopore sequencing for characterizing unexpected complex structural alterations caused by genome editing.