Brain-Computer Interface

Launched in 2017 to widespread publicity due to the involvement of tech magnate and outspoken futurist Elon Musk, Neuralink Corp. aims to develop an advanced brain-computer interface (BCI) platform capable of assisting in the treatment of serious neurological conditions with longer-term goals of approaching transhumanism through nonmedical human enhancement to enable human-machine “symbiosis with artificial intelligence.” The first published description of a complete prototype Neuralink system, detailed by Muskin the company's only white paper to date, describes a closed-loop, invasive BCI architecture with an unprecedented magnitude of addressable electrodes. Invasive BCI systems require surgical implantation to allow for directly targeted capture and/or stimulation of neural spiking activity in functionally associated clusters of neurons beneath the surface of the cortex.

Leukemic Cells Expressing ETV6-Frk Identified in a Refractory B-ALL Patient Are Sensitive to Dasatinib in Vitro

[Background] ETV6-FRK is a rare kinase-related fusion gene which was identified only in acute myeloid leukemia (Hosoya N, et al. Genes Chromosomes Cancer. 2005). Herein, we firstly identified ETV6-FRK fusion gene in a patient with high-risk pediatric B cell precursor ALL (B-ALL). Because FRK is Src family tyrosine kinase, we performed functional analysis of ETV6-FRK to establish molecular targeting therapy. [Patient] A 11-year-old boy with B-ALL was refractory to conventional chemotherapy and received allogeneic bone marrow transplantation (allo-BMT) following two courses of blinatumomab. This patient maintains complete remission for three months after allo-BMT. Cytogenetic analysis demonstrated t(6;12)(q21;p13) as a part of complex karyotype. Targeted capture mRNA sequencing identified ETV6-FRK fusion transcript in this patient. [Materials and methods] ETV6-FRK fusion was validated by RT-PCR of the diagnostic leukemic sample from this patient. Full length of ETV6-FRK cDNA was cloned into retroviral vector with Tet-On system. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing ETV6-FRK (Ba/F3-ETV6-FRK) under doxycycline (DOX) dependent manner. Ba/F3-ETV6-FRK was analyzed whether IL-3 independent growth was achieved. To determine whether aberrant activation of FRK-STAT pathway as the downstream effects of ETV6-FRK, activation of FRK-STAT pathway was evaluated by western blot. Finally, in cytotoxic assay, proliferation of Ba/F3-ETV6-FRK was assessed under the media with various concentrations of dasatinib, a tyrosine kinase inhibitor. [Results and discussions] Sequencing of RT-PCR product revealed that ETV6 exon 4 was fused in-frame to FRK exon 3, creating an ETV6-FRK fusion gene. The ETV6-FRK fusion gene produced a chimeric protein consisting of the entire pointed (PNT) oligomerization domain of ETV6 and the kinase domain of FRK (Fig 1). The expression of ETV6-FRK in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-ETV6-FRK proliferated without IL-3 in contrast to Ba/F3 cells not expressing ETV6-FRK (p<0.01), suggesting ETV6-FRK had proliferation activity. Western blot analysis revealed constitutive phosphorylation of tyrosine residues of ETV6-FRK and STAT5/STAT3/STAT1, suggesting constitutive activation of FRK-STATs pathway was associated with IL-3 independent proliferation activity of ETV6-FRK. Considering that dasatinib, which is Src-kinase inhibitor, could block constitutive phosphorylation of ETV6-FRK, we hypothesized that dasatinib might block the IL-3 independent proliferation of Ba/F3-ETV6-FRK. In vitro killing assay showed that dasatinib suppressed efficiently the proliferation of Ba/F3-ETV6-FRK with 50% inhibitory concentration (IC50) 1.64 ± 0.02 nM, although dasatinib didn't show any effect on Ba/F3 cells not expressing ETV6-FRK (Fig. 2). Annexin V assay determined that 35.8 ± 6.9 % of Ba/F3-ETV6-FRK with dasatinib (10nM, 48hrs) were apoptotic than Ba/F3 cells not expressing ETV6-FRK (Fig. 3, p<0.01). These findings suggested that dasatinib abolished the proliferation activity of ETV6-FRK selectively. [Conclusion] We identified the first patient of pediatric high-risk B-ALL harboring ETV6-FRK fusion by targeted capture mRNA sequencing, who was refractory to the conventional chemotherapy. We also provide the first evidence that dasatinib could abrogate proliferation activity of ETV6-FRK in vitro, suggesting that dasatinib might be effective for the patient with B-ALL carrying a ETV6-FRK fusion. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Der(1;7)(q10;p10) Presents with a Unique Genetic Profile and Frequent ETNK1 Mutations in Myeloid Neoplasms

Background Der(1;7)(q10;p10) (der(1;7) is an unbalanced translocation recurrently found in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and related disorders. Caused by a recombination between two homologous alphoid sequencing D1Z7 and D7Z1 on chromosomes 1 and 7, respectively, it results in monosomy 7q and trisomy 1q, which is implicated in the pathogenesis of der(1;7)-positive myeloid neoplasms. Previous studies reported frequent co-occurrence of +8 and del(20q), as well as RUNX1 mutations, the genetic and clinical characteristics of this abnormality has not fully been elucidated. Methods In this study, we enrolled a total of 153 cases myeloid neoplasms positive for der(1;7) from Japanese and German cohorts, in which co-occurring genetic lesions were analyzed using whole exome and/or targeted-capture sequencing. An additional 3,223 MDS and related neoplasm cases were also analyzed using targeted-capture sequencing to identify der(1;7)-specific genomic features. Results Ethnicity was evaluated comparing the frequency of der(1;7) in 944 German MDS cases and 763 Japanese MDS cases. Der(1;7) cases were observed at a higher frequency in Japanese MDS cohort compared to German MDS cohort (73/763 cases versus 4/944 cases, p < 0.00001). Der(1;7) cases showed a strong male predominance (86.3%) (p<0.001). Of 153 myeloid neoplasm patients harboring der(1;7), 114 were diagnosed with MDS, 28 with AML, 5 with MDS-MPN and 1 with MPN. Targeted-capture sequencing revealed mutations in common myeloid drivers (n=61) in 96% of der(1;7) cases. The most frequently mutated gene was RUNX1 with 46%, followed by ETNK1 (24.5%) and EZH2 (24.5%). Of interest, ETNK1 mutation was identified as the most unique to der(1;7) when compared to myeloid neoplasm cases without der(1;7) (n=3,066) [odds ratio (OR)=15.06], followed by ETV6 (OR=9.35) and EZH2 (OR=6.52). To further examine the uniqueness of this mutation profile, the mutational profile of der(1;7) was compared to those myeloid neoplasm cases harboring amp(1q) (n=52) and monosomy 7 (n=105). Highly frequent ETV6 and ETNK1 mutations were highly unique to der(1;7) cases when compared to amp(1q) cases (OR=3.72, OR=2.57, respectively). BCOR and ETNK1 mutations were highly unique to der(1;7) cases when compared to monosomy 7 cases (OR=35.88, OR=4.29, respectively). Both amp(1q) and monosomy 7 cases showed a higher mutation rate in TP53 compared to der(1;7) cases (49.1% and 51%, respectively, vs 3.5 %) . From these mutational characteristics, ETNK1 was identified as being the most unique to der(1;7) when compared to amp(1q), monosomy 7 and other myeloid neoplasm cases. ETNK1-mutated der(1;7) cases were featured with eosinophilia (p < 0.0005), a lack of RAS pathway mutations and trisomy 8 when compared to ETNK1-wild type der(1;7) cases. Survival analysis was conducted to elucidate the difference in survival in der(1;7) cases (n=65) versus myeloid neoplasm cases (n=2066). Der(1;7)-harboring myeloid neoplasm cases had a median overall survival of 6.8 months (95% CI, 3.5 to 11.9) and non-der(1;7) harboring myeloid neoplasm cases were 11.8 months (95% CI, 10.5 to 12.6). Thus, der(1;7)-harboring myeloid neoplasm cases had poorer prognosis (p<0.001). Conclusion In conclusion, der(1;7) is an unbalanced translocation that occurs predominantly in males and is seen more frequently in Japanese than Caucasian populations. Der(1;7) cases present with a mutational profile that is distinct from other myeloid neoplasm cases such as those with amp(1q) and monosomy7/del(7q), showing frequency of ETNK1 mutations. Disclosures Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Atsuta: Astellas Pharma Inc.: Speakers Bureau; Mochida Pharmaceutical Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Kyowa Kirin Co., Ltd: Honoraria; Meiji Seika Pharma Co, Ltd.: Honoraria. Handa: Ono: Honoraria; BMS: Honoraria; Janssen: Honoraria; Daiichi Sankyo: Research Funding; Celgene: Honoraria, Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Honoraria; MSD: Research Funding; Shionogi: Research Funding. Ohyashiki: Novartis Pharma: Other: chief clinical trial; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Ogawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company.

Overcoming limitations to environmental DNA studies: A coastal temperate reference sequence database for multiple chloroplast gene regions generated in a single assay.

A proliferation in environmental DNA (eDNA) research has increased the reliance on reference sequence databases to assign unknown DNA sequences to known taxa. Without comprehensive reference databases, DNA extracted from environmental samples cannot be correctly assigned to taxa, limiting the use of this genetic information to identify organisms in unknown sample mixtures. For animals, standard metabarcoding practices involve amplification of the mitochondrial Cytochrome-c oxidase subunit 1 (CO1) region, which is a universally amplifyable region across majority of animal taxa. This region, however, does not work well as a DNA barcode for plants and fungi, and there is no similar universal single barcode locus that has the same species resolution. Therefore, generating reference sequences has been more difficult and several loci have been suggested to be used in parallel to get to species identification. For this reason, we developed a multi-gene targeted capture approach to generate reference DNA sequences for plant taxa across 20 target chloroplast gene regions in a single assay. We successfully compiled a reference database for 93 temperate coastal plants including seagrasses, mangroves, and saltmarshes/samphire’s. We demonstrate the importance of a comprehensive reference database to prevent species going undetected in eDNA studies. We also investigate how using multiple chloroplast gene regions impacts the ability to discriminate between taxa.

Exogene: A performant workflow for detecting viral integrations from paired-end next-generation sequencing data

The integration of viruses into the human genome is known to be associated with tumorigenesis in many cancers, but the accurate detection of integration breakpoints from short read sequencing data is made difficult by human-viral homologies, viral genome heterogeneity, coverage limitations, and other factors. To address this, we present Exogene, a sensitive and efficient workflow for detecting viral integrations from paired-end next generation sequencing data. Exogene’s read filtering and breakpoint detection strategies yield integration coordinates that are highly concordant with long read validation. We demonstrate this concordance across 6 TCGA Hepatocellular carcinoma (HCC) tumor samples, identifying integrations of hepatitis B virus that are also supported by long reads. Additionally, we applied Exogene to targeted capture data from 426 previously studied HCC samples, achieving 98.9% concordance with existing methods and identifying 238 high-confidence integrations that were not previously reported. Exogene is applicable to multiple types of paired-end sequence data, including genome, exome, RNA-Seq and targeted capture.

A hybrid capture RNA bait set for resolving genetic and evolutionary relationships in angiosperms from deep phylogeny to intraspecific lineage hybridization

Novel multi-gene targeted capture probes have been developed with the objective of obtaining multi-locus high quality sequence reads across any angiosperm lineage. Using existing genomic and transcriptomic data, two independent single assay probe/bait sets have been developed, the first targeting conserved exons from 20 low copy nuclear genes (OzBaits_NR V1.0) and the second, 19 plastid gene regions (OZBaits_CP V1.0). These universal bait sets can efficiently generate DNA sequence data that are suitable for systematics and evolutionary studies of flowering plants. The bait sets can be ordered as Daicel-Arbor Sciences custom myBaits. We demonstrate the utility of the bait set in consistently recovering the targeted genomic regions across an evolutionarily broad range of angiosperm taxa.

CACNA1A-p.Thr501Met mutation associated with familial hemiplegic migraine: a family report

Background and aims Hemiplegic migraine (HM) is a rare form of migraine characterized by the presence of a motor and other types of aura. HM can be sporadic or familial. Familial hemiplegic migraine (FHM) is an autosomal dominant disorder, classified into 3 subtypes, based on the gene involved (CACNA1A in FHM1, ATP1A2 in FHM2 and SCN1A in FHM3). The clinical presentation is highly heterogeneous and some attacks may be severe. We report the clinical characteristics and genetic analysis of 12 patients belonging to a family with CACNA1A-p.Thr501Met gene mutation. Methods We screened for mutations in CACNA1A gene 15 patients belonging to the same family. The exonic sequences of CACNA1A were analyzed using a Tru-seq® Custom Amplicon (TSCA) (Illumina Inc., San Diego, CA) targeted capture and paired end library kit. Sanger sequencing was used to confirm CACNA1A variants and segregation analysis. Results CACNA1A-p.Thr501Met mutation was found in 12 of the 15 patients screened, which was compatible with the diagnosis of FHM1. Attacks of hemiplegic migraine were reported by 10 of the 12 subjects (83.33%). Only one subject developed persistent mild cerebellar symptoms and none of the subjects developed cerebellar atrophy. Discussion The variant p.Thr501Met was described previously in association with episodic ataxia and rarely with FHM related to cerebellar symptoms. FHM1 has a broad clinical spectrum and about half of the families have cerebellar involvement. In our study, only one patient developed persistent cerebellar deficits. These data suggest that CACNA1A-p.Thr501Met mutation can occur prevalently as hemiplegic migraine.

The Potential and Analysis of ctDNA Sequencing in Hepatocellular Carcinoma

Background: The Genome map of hepatocellular carcinoma (HCC) is complex. We used the next generation of targeted sequencing technology to decipher the mutations in patients with liver cancerMethods: The circulating tumor cell DNA (ctDNA) of 10 patients with hepatocellular carcinoma (Including 8 cases of primary hepatocellular carcinoma and 2 cases of metastatic hepatocellular carcinoma) were sequenced. We used SAMtools to detect and screen single nucleotide polymorphism (SNP) and insertion deletion (INDEL) mutations, and ANNOVAR to annotate the structure and function of the detected mutations.Results: Targeted capture and deep sequencing of 560 cancer-related genes in 10 liver cancer ctDNA samples showed 8950 single nucleotide variations (SNVS) mutations and 70 INDELS. The most common mutation gene was PDE4DIP, followed by SYNE1, KMT2C, PKHD1 and FN1. According to the American College of Medical Genetics and Genomics (ACMG) guidelines, we authenticated 54 pathogenic and possible pathogenic mutations in 39 genes in exons and splice regions of 10 patients with HCC.Conclusion: Our research provides the gene mutation map of Chinese hepatocarcinoma patients, and enriches the understanding of the pathogenesis of HCC.

A multi-gene region targeted capture approach to detect plant DNA in environmental samples: A case study from coastal environments

Metabarcoding of plant DNA recovered from environmental samples, termed environmental DNA (eDNA), has been used to detect invasive species, track biodiversity changes and reconstruct past ecosystems. The P6 loop of the trnL intron is the most widely utilized gene region for metabarcoding plants due to the short fragment length and subsequent ease of recovery from degraded DNA, which is characteristic of environmental samples. However, the taxonomic resolution for this gene region is limited, often precluding species level identification. Additionally, targeting gene regions using universal primers can bias results as some taxa will amplify more effectively than others. To increase the ability of DNA metabarcoding to better resolve flowering plant species (angiosperms) within environmental samples, and reduce bias in amplification, we developed a multi-gene targeted capture method that simultaneously targets 20 chloroplast gene regions in a single assay across all flowering plant species. Using this approach, we effectively recovered multiple chloroplast gene regions for three species within artificial DNA mixtures down to 0.001 ng/uL of DNA. We tested the detection level of this approach, successfully recovering target genes for 10 flowering plant species. Finally, we applied this approach to sediment samples containing unknown compositions of environmental DNA and confidently detected plant species that were later verified with observation data. Targeting multiple chloroplast gene regions in environmental samples enabled species-level information to be recovered from complex DNA mixtures. Thus, the method developed here, confers an improved level of data on community composition, which can be used to better understand flowering plant assemblages in environmental samples.