Somatic Deletions Research Articles

Mitochondria DNA copy number, mitochondria DNA total somatic deletions, Complex I activity, synapse number, and synaptic mitochondria number are altered in schizophrenia and bipolar disorder

Mitochondrial dysfunction is a neurobiological phenomenon implicated in the pathophysiology of schizophrenia and bipolar disorder that can synergistically affect synaptic neurotransmission. We hypothesized that schizophrenia and bipolar disorder share molecular alterations at the mitochondrial and synaptic levels. Mitochondria DNA (mtDNA) copy number (CN), mtDNA common deletion (CD), mtDNA total deletion, complex I activity, synapse number, and synaptic mitochondria number were studied in the postmortem human dorsolateral prefrontal cortex (DLPFC), superior temporal gyrus (STG), primary visual cortex (V1), and nucleus accumbens (NAc) of controls (CON), and subjects with schizophrenia (SZ), and bipolar disorder (BD). The results showed (i) the mtDNA CN is significantly higher in DLPFC of both SZ and BD, decreased in the STG of BD, and unaltered in V1 and NAc of both SZ and BD; (ii) the mtDNA CD is significantly higher in DLPFC of BD while unaltered in STG, V1, and NAc of both SZ and BD; (iii) The total deletion burden is significantly higher in DLPFC in both SZ and BD while unaltered in STG, V1, and NAc of SZ and BD; (iv) Complex I activity is significantly lower in DLPFC of both SZ and BD, which is driven by the presence of medications, with no alteration in STG, V1, and NAc. In addition, complex I protein concentration, by ELISA, was decreased across three cortical regions of SZ and BD subjects; (v) The number of synapses is decreased in DLPFC of both SZ and BD, while the synaptic mitochondria number was significantly lower in female SZ and female BD compared to female controls. Overall, these findings will pave the way to understand better the pathophysiology of schizophrenia and bipolar disorder for therapeutic interventions.

Proteogenomic Markers of Chemotherapy Resistance and Response in Triple-Negative Breast Cancer.

Proteogenomic analysis of triple-negative breast tumors revealed a complex landscape of chemotherapy response associations, including a 19q13.31-33 somatic deletion encoding genes serving lagging-strand DNA synthesis (LIG1, POLD1, and XRCC1), that correlate with lack of pathologic response, carboplatin-selective resistance, and, in pan-cancer studies, poor prognosis and CIN. This article is highlighted in the In This Issue feature, p. 2483.

Recurrent NOMO1 Gene Deletion Is a Potential Clinical Marker in Early-Onset Colorectal Cancer and Is Involved in the Regulation of Cell Migration.

Simple SummaryThe incidence of EOCRC (age < 50 years at diagnosis) with unknown causes is rising worldwide, necessitating the mechanistical analysis of its molecular basis. The NOMO1 gene is deleted in a high number of EOCRC tumors compared to LOCRC. In this work, we aimed to test the NOMO1 gene mutational profile in EOCRC tumors and to characterize the effect of NOMO1 loss in different CRISPR/cas9-edited cell lines, as well as in murine models. Here, we show that the NOMO1 gene can be inactivated not only by deletion but also by pathogenic mutations in EOCRC. Our results indicate that NOMO1 loss could be a passenger mutation in the development of EOCRC, although it contributes significantly to colon cancer cell migration.The incidence of early-onset colorectal cancer (EOCRC; age younger than 50 years) has been progressively increasing over the last decades globally, with causes unexplained. A distinct molecular feature of EOCRC is that compared with cases of late-onset colorectal cancer, in EOCRC cases, there is a higher incidence of Nodal Modulator 1 (NOMO1) somatic deletions. However, the mechanisms of NOMO1 in early-onset colorectal carcinogenesis are currently unknown. In this study, we show that in 30% of EOCRCs with heterozygous deletion of NOMO1, there were pathogenic mutations in this gene, suggesting that NOMO1 can be inactivated by deletion or mutation in EOCRC. To study the role of NOMO1 in EOCRC, CRISPR/cas9 technology was employed to generate NOMO1 knockout HCT-116 (EOCRC) and HS-5 (bone marrow) cell lines. NOMO1 loss in these cell lines did not perturb Nodal pathway signaling nor cell proliferation. Expression microarrays, RNA sequencing, and protein expression analysis by LC–IMS/MS showed that NOMO1 inactivation deregulates other signaling pathways independent of the Nodal pathway, such as epithelial–mesenchymal transition and cell migration. Significantly, NOMO1 loss increased the migration capacity of CRC cells. Additionally, a gut-specific conditional NOMO1 KO mouse model revealed no subsequent tumor development in mice. Overall, these findings suggest that NOMO1 could play a secondary role in early-onset colorectal carcinogenesis because its loss increases the migration capacity of CRC cells. Therefore, further study is warranted to explore other signalling pathways deregulated by NOMO1 loss that may play a significant role in the pathogenesis of the disease.

A naturally arising broad and potent CD4-binding site antibody with low somatic mutation.

The induction of broadly neutralizing antibodies (bNAbs) is a potential strategy for a vaccine against HIV-1. However, most bNAbs exhibit features such as unusually high somatic hypermutation, including insertions and deletions, which make their induction challenging. VRC01-class bNAbs not only exhibit extraordinary breadth and potency but also rank among the most highly somatically mutated bNAbs. Here, we describe a VRC01-class antibody isolated from a viremic controller, BG24, that is much less mutated than most relatives of its class while achieving comparable breadth and potency. A 3.8-Å x-ray crystal structure of a BG24-BG505 Env trimer complex revealed conserved contacts at the gp120 interface characteristic of the VRC01-class Abs, despite lacking common CDR3 sequence motifs. The existence of moderately mutated CD4-binding site (CD4bs) bNAbs such as BG24 provides a simpler blueprint for CD4bs antibody induction by a vaccine, raising the prospect that such an induction might be feasible with a germline-targeting approach.

Absence of NOTCH1 mutation and presence of CDKN2A deletion predict progression of esophageal lesions.

Currently, surveillance for esophageal squamous cell carcinoma (ESCC) runs a risk of underestimation of early lesions which show absence of iodine staining, with no or only mild histologic changes. The development of molecular markers that indicate risk of progression is thus warranted. We performed whole-exome sequencing on biopsies from two sequential endoscopies of a single esophageal lesion and matching blood samples. There were 27 pairs of age-, gender-, pathologic stage-, and sampling interval-matched progressors and non-progressors identified in a prospective community-based ESCC screening trial. Putative molecular progression markers for ESCC were first evaluated by comparing somatic mutation, copy number alteration (CNA), and mutational signature information among progressors and non-progressors. These markers were then validated with another 24 pairs of matched progressors and non-progressors from the same population using gene alteration status identified by target sequencing and quantitative PCR. Progressors had more somatic mutation and CNA burden, as well as apolipoprotein B mRNA editing catalytic polypeptide-like and age-related signature weights compared with non-progressors. A gene score consisting of somatic NOTCH1 mutation and CDKN2A deletion is predictive of risk of progression in lesions which show absence of iodine staining under endoscopy but have no or only mild dysplasia. This gene score was also validated in an external cohort of matched progressors and non-progressors. Absence of NOTCH1 mutation and presence of CDKN2A deletion are markers of progression in squamous lesions of the esophagus. This gene score would be an ideal indicator for assisting the pathologist in the identification of high-risk individuals who could be potentially 'missed' or subject to a risk underestimation by histologic analysis, and might improve the performance of ESCC surveillance. © 2022 The Pathological Society of Great Britain and Ireland.

Germline variants of ATG7 in familial cholangiocarcinoma alter autophagy and p62

Autophagy is a housekeeping mechanism tasked with eliminating misfolded proteins and damaged organelles to maintain cellular homeostasis. Autophagy deficiency results in increased oxidative stress, DNA damage and chronic cellular injury. Among the core genes in the autophagy machinery, ATG7 is required for autophagy initiation and autophagosome formation. Based on the analysis of an extended pedigree of familial cholangiocarcinoma, we determined that all affected family members had a novel germline mutation (c.2000C>T p.Arg659* (p.R659*)) in ATG7. Somatic deletions of ATG7 were identified in the tumors of affected individuals. We applied linked-read sequencing to one tumor sample and demonstrated that the ATG7 somatic deletion and germline mutation were located on distinct alleles, resulting in two hits to ATG7. From a parallel population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G p.Asp522Glu (p.D522E)) associated with increased risk of cholangiocarcinoma. To characterize the impact of these germline ATG7 variants on autophagy activity, we developed an ATG7-null cell line derived from the human bile duct. The mutant p.R659* ATG7 protein lacked the ability to lipidate its LC3 substrate, leading to complete loss of autophagy and increased p62 levels. Our findings indicate that germline ATG7 variants have the potential to impact autophagy function with implications for cholangiocarcinoma development.

Abstract 2628: Exploring synthetic lethality between VPS4A and VPS4B paralog enzymes in pancreatic and colon cancer

Abstract Tumor suppressors can be inactivated through somatic gene deletions in cancer. Loss of the tumor suppressor SMAD4 is associated with 18q21 chromosomal deletion and frequently observed in pancreatic and colon cancers. The large-scale cancer dependency Broad and Sanger lethality screens revealed a synthetic lethal relationship between genomic loss of SMAD4 and vacuolar protein sorting 4 homolog A (VPS4A) with selectivity in pancreatic and colon cancer cell lines. VPS4A is a functional paralog to vacuolar protein sorting 4 homolog B (VPS4B), where VPS4B is located on 18q21 and correlates with SMAD4 copy number. VPS4A and VPS4B are paralog ATPases that function in the endosomal sorting complex required for vesicular transport and budding, which is required for cellular homeostasis and survival. As a consequence, collateral loss of VPS4B associated with SMAD4 loss results in a selective sensitivity for VPS4A deletion. Therefore, VPS4A is a possible therapeutic target in tumors deficient for VPS4B.We investigated the paralog synthetic lethality relationship between VPS4A and VPS4B in pancreatic and colon cancer cell lines. We rescued cell growth using ectopic wildtype VPS4A expression following combined knockdown of VPS4A and VPS4B. We further investigated if the ATPase activity of VPS4A was required to rescue paralog lethality using ectopic expression of mutant catalytically-dead VPS4A proteins. We discovered that the ATPase activity of VPS4A was required to rescue paralog lethality, as expression of the catalytically-dead VPS4A proteins did not support cell growth. However, it was noted that expression of the mutant proteins resulted in cell growth attenuation in addition to the inability to rescue. To investigate this observation, we expressed mutant and wild-type VPS4A proteins in cell lines without knock-down of endogenous protein. We found expressing either of the mutant VPS4A proteins, but not wild-type VPS4A protein, decreased cell growth in the presence of the endogenous protein. Decreased cell growth with the expression of the ATPase catalytic mutants suggest a potential dominant negative effect. This negative effect on cell growth with an ATPase inactive protein has the potential to translate to the effect observed with a selective VPS4A ATPase inhibitor. Additionally, the discovery of a selective ATPase inhibitor against VPS4A or VPS4B may be challenging due to the high sequence similarity within the ATPase domain. However, the microtubule interacting and trafficking (MIT) domain between VPS4A and VPS4B shows less sequence similarity and has a higher likelihood for selectivity. In summary, we confirmed the paralog lethality relationship between VPS4A and VPS4B and the dependence on the ATPase activity of VPS4A. However, the selective targeting of the VPS4A ATPase activity may result in unintended cell growth impairment, the lack of synthetic lethality, and potential for adverse toxicity. Citation Format: Timothy Nacarelli, Nelisa Bechtel, Yanzhe Gao, Kurt Auger. Exploring synthetic lethality between VPS4A and VPS4B paralog enzymes in pancreatic and colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2628.

MEDB-15. Dynamic chromatin alteration induces oncogenic hijacking by essential transcriptional factors during SHH medulloblastoma tumorigenesis

Medulloblastoma is a malignant brain tumor that occurs in the cerebellum, most frequently in children. Medulloblastoma is molecularly classified into four major groups, and therapies are now being developed according to the nature of these groups and subgroups. However, there are currently no effective molecularly targeted drugs for most of these groups. In recent years, we have been analyzing the genomes of medulloblastomas to identify genetic mutations involved in tumorigenesis. Among them, mutations in chromatin modifiers are frequently detected in medulloblastoma, suggesting the importance of alterations in the epigenome in tumor development. However, it remains unclear how epigenomic changes are involved in tumorigenesis. Here, we have used the SHH-group medulloblastoma (MB-SHH) mouse model to understand the epigenomic changes during tumor development and identify new therapeutic targets for medulloblastoma. To this end, we utilized an Atoh1-EGFP:Ptch1+/- mice that specifically label cerebellar granule cell progenitors (GNPs) that are known to be the cellular origin of MB-SHH, preneoplastic (PNCs) and tumor cells with EGFP during MB-SHH formation. Following FACS-based collection of EGFP-positive cells, comparative ATAC-seq analysis revealed that the open chromatin regions dynamically changed during transformation of GNPs into PNCs with enrichment of nuclear factor I (NFI) binding motifs. Cut & Tag analysis on these cells showed that NFI proteins bound chromatin regions that become more accessible during tumorigenesis, suggesting that NFI could play an important role in tumorigenesis after the epigenomic changes. Indeed, CRISPR-engineered in vivo somatic deletion of Nfia and/or Nfib prevented hyperplasia formation, confirming their essential role in tumor initiation. Knockdown of either NFIA or NFIB in patient-derived xenograft models also attenuated tumor growth. Thus, our study has uncovered a novel oncogenic mechanism that contributes to the development of MB-SHH tumors via alterations of accessible chromatin and aberrant DNA binding of NFI transcription factors.

Learning the statistics and landscape of somatic mutation-induced insertions and deletions in antibodies.

Affinity maturation is crucial for improving the binding affinity of antibodies to antigens. This process is mainly driven by point substitutions caused by somatic hypermutations of the immunoglobulin gene. It also includes deletions and insertions of genomic material known as indels. While the landscape of point substitutions has been extensively studied, a detailed statistical description of indels is still lacking. Here we present a probabilistic inference tool to learn the statistics of indels from repertoire sequencing data, which overcomes the pitfalls and biases of standard annotation methods. The model includes antibody-specific maturation ages to account for variable mutational loads in the repertoire. After validation on synthetic data, we applied our tool to a large dataset of human immunoglobulin heavy chains. The inferred model allows us to identify universal statistical features of indels in heavy chains. We report distinct insertion and deletion hotspots, and show that the distribution of lengths of indels follows a geometric distribution, which puts constraints on future mechanistic models of the hypermutation process.

NOTCH4 mutation as a potential predictive biomarker for immunotherapy in NRAS wildtype melanoma.

e21594 Background: NRAS wildtype melanoma account for 80-85% melanoma patients and have been reported had a better response for immunotherapy. Although PD-L1 expression and tumor mutation burden (TMB) have been considered as important biomarkers for immunotherapy in many types of tumors, the predictive efficacy of them in melanoma is controversial. Since evidences showed NOTCH pathway mutation was a potential predictive biomarker for immune checkpoint inhibitors in some types of tumors. Here we explored the relationship between NOTCH family genes mutations and efficacy of NRAS wildtype melanoma immunotherapy. Methods: 265 NRAS wildtype ICI-pretreatment melanomas from cBioPortal (https://www.cbioportal.org), which is divided to discovery (N = 76) and validation (N = 189) cohort to analyze the association between NOTCH family genes (NOTCH1-4) mutation and efficacy of ICIs therapy. All nonsynonymous somatic mutations, including nonsense, missense, nonstop, frameshift deletion and insertion, in-frame deletion and insertion, and splice site mutations were considered for inclusion in our study. The potential mechanism was subsequently explored through RNA expression in immunotherapy treated population. Results: Patients with NOTCH4-Mut were identified to be associated with prolonged overall survival (OS) in discovery (HR: 0.30, 95%CI: 0.11-0.83, P = 0.01) and validation cohort (HR: 0.21, 95%CI: 0.07-0.68, P = 0.003). Further exploration found that NOTCH4-Mut tumors had higher tumor mutation burden (TMB) and tumor neoantigen burden (TNB) (P &lt; 0.05). RNA data revealed that enhanced anti-tumor immunity was observed in NOTCH4-Mut tumors. Activated CD4 memory T cell, CD8 T cell, follicular helper T cell (Tfh) and Neutrophils were more abundant in NOTCH4-Mut tumors. In addition, the results of enrichment analysis showed that several immune-related pathways varied significantly between NOTCH4-Mut and NOTCH4-Wt tumors, including Interferon γ response, DNA repair, epithelial mesenchymal transition (EMT), angiogenesis. Conclusions: NOTCH4 mutation may promote tumor immunity and serve as a biomarker to predict good immune response in NRAS wildtype melanoma and guiding immunotherapeutic responsiveness.

Identification of distinct genomic features reveals frequent somatic AHNAK and PTEN mutations predominantly in primary malignant melanoma presenting in the ureter.

Primary malignant melanoma of the ureter is extremely rare. Genetic variants to the increased risk of developing the disease have not yet been investigated. Tumour mutation profiling for primary malignant melanoma of the ureter was performed by whole-exome sequencing. Immunohistochemistry was performed to verify histopathological features and the variants of predisposing genes and driver mutation genes. Furthermore, we conducted a literature review and Surveillance, Epidemiology and End Result-based study by searching public databases. We identified 38 somatic single nucleotide variants and 9 somatic insertions and deletions (INDELs) in tumour specimens. After filtering with the Cancer Gene Census database, seven predisposing genes and two driver mutation genes were identified. Moreover, the immunohistochemical profile showed that tumour cells were positive for Melan-A, melanoma gp100 human melanoma black 45 (HMB45), S100 beta and P53. The expression levels of two driver mutation genes (phosphatase and tensin homolog (PTEN) and desmoyokin (AHNAK) and five predisposing genes (AT-rich interaction domain 1B (ARID1B), catalase, eukaryotic translation initiation factor 4 gamma 3 (EIF4G3), ANK3 and collagen type I) were significantly downregulated in tumour tissues compared to paracancerous tissues. In the literature review and Surveillance, Epidemiology and End Results-based study, patients with primary malignant melanoma of the urinary tract had worse clinical outcomes than patients with primary urothelial carcinoma after 1:2 propensity score matching (P=0.010). Additionally, Cox multivariate analysis for patients with primary malignant melanoma of the urinary tract indicated that distant metastasis (hazard ratio=1.185; P=0.044) was an independent predictor for overall survival, and tumour focality (hazard ratio=0.602; P=0.017) and non-surgery (hazard ratio=0.434; P=0.003) were independent factors for tumour progression. Our study is the first to provide evidence that the distinct phenotypes of primary malignant melanoma of the ureter may be due to different genetic variations. The prognosis of primary malignant melanoma of the urinary tract was poorer than that of primary urothelial carcinoma of the urinary tract.

Lung metastases and subsequent malignant transformation of a fumarate hydratase -deficient uterine leiomyoma

Uterine leiomyomas, or fibroids, are very common smooth muscle tumors. Their potential to metastasize or transform into leiomyosarcomas is extremely low. Here, we report a patient who underwent hysterectomy due to a large leiomyoma and who was diagnosed with pulmonary tumors seven and nine years later. Histopathological re-evaluation confirmed the cellular leiomyoma diagnosis for the uterine tumor, whereas the pulmonary tumors met the diagnostic criteria of a leiomyosarcoma. Whole-exome sequencing revealed very similar mutational profiles in all three tumors, including a somatic homozygous deletion in a rare, but well-established leiomyoma driver gene FH. Tumor evolution analysis confirmed the clonal origin of all three tumors. In addition to mutations shared by all three tumors, pulmonary tumors harbored additional alterations affecting e.g. the cancer-associated genes NRG1 and MYOCD. The second pulmonary leiomyosarcoma harbored additional changes, including a mutation in FGFR1. In global gene expression profiling, the uterine tumor showed similar expression patterns as other FH-deficient leiomyomas. Taken together, this comprehensive molecular data supports the occasional metastatic capability and malignant transformation of uterine leiomyomas. Further studies are required to confirm whether FH-deficient tumors and/or tumors with cellular histopathology have higher malignant potential than other uterine leiomyomas.

Novel Pathogenic Sequence Variation m.5789T>C Causes NARP Syndrome and Promotes Formation of Deletions of the Mitochondrial Genome.

Background and ObjectivesWe report the pathogenic sequence variant m.5789T>C in the anticodon stem of the mitochondrial tRNA for cysteine as a novel cause of neuropathy, ataxia, and retinitis pigmentosa (NARP), which is usually associated with pathogenic variants in the MT-ATP6 gene.MethodsTo address the correlation of oxidative phosphorylation deficiency with mutation loads, we performed genotyping on single laser-dissected skeletal muscle fibers. Stability of the mitochondrial tRNACys was investigated by Northern blotting. Accompanying deletions of the mitochondrial genome were detected by long-range PCR and their breakpoints were determined by sequencing of single-molecule amplicons.ResultsThe sequence variant m.5789T>C, originating from the patient's mother, decreases the stability of the mitochondrial tRNA for cysteine by disrupting the anticodon stem, which subsequently leads to a combined oxidative phosphorylation deficiency. In parallel, we observed a prominent cluster of low-abundance somatic deletions with breakpoints in the immediate vicinity of the m.5789T>C variant. Strikingly, all deletion-carrying mitochondrial DNA (mtDNA) species, in which the corresponding nucleotide position was not removed, harbored the mutant allele, and none carried the wild-type allele.DiscussionIn addition to providing evidence for the novel association of a tRNA sequence alteration with NARP syndrome, our observations support the hypothesis that single nucleotide changes can lead to increased occurrence of site-specific mtDNA deletions through the formation of an imperfect repeat. This finding might be relevant for understanding mechanisms of deletion generation in the human mitochondrial genome.

EP318: Concurrent somatic 9p21 deletion and germline EP300 variant in a patient with T-ALL, immunodeficiency and malformations: Phenotypic expansion or comorbidities?

EP300 gene encodes E1A-associated protein p300 that function as co-activators of transcription and regulates transcription via chromatin remodeling. Pathogenic variants in EP300 on chromosome 22q13.2 have been reported in about 5–10% of individuals with Rubinstein-Taybi syndrome (RTS2) [OMIM# 613684], as well as in few patients with Menke-Hennekam syndrome (MKHK2) [OMIM# 618333]. In addition, it may function as a potential oncogene as somatic variants in EP300 have been reported in various cancers with high prevalence such as skin squamous cell carcinoma, marginal zone B-cell lymphoma, bladder carcinoma, and follicular lymphoma.

Hypoxia-induced macropinocytosis represents a metabolic route for liver cancer

Hepatocellular carcinoma (HCC) invariably exhibits inadequate O2 (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive.

The affinity maturation, characteristics and application of HIV-1 broadly neutralizing antibodies

Hundreds of broadly neutralizing antibodies(bNAbs) were successfully isolated from long-term nonprogression(LTNP) of human immunodeficiency virus type 1(HIV-1) infected individuals. Some bNAbs were illustrated could reduce the viral load and the risk of HIV-1 infection. Today, HIV-1 bNAbs are at the center of vaccine development and passive immunization treatment. Usually, the activity of neutralizing antibodies depends on the epitope. The affinity of neutralizing antibodies also plays a vital role in its inhibitory effect. Multiple affinity maturation in vivo actually provides the broad and potent neutralizing activity of HIV-1 bNAbs. When high affinity HIV-1 bNAbs applied in clinic, it can help immune system to remove virus with lower dosage and fewer side effect. While affinity maturation, HIV-1 bNAbs shows unique characteristics, such as extensive of somatic hypermutation(SHM), in-frame insertion and deletion and long CDR 3 region of heavy chain. The key points in the progress that HIV-1 bNAbs affinity maturation will help us understand the relationship between antibodies neutralizing capability and its characteristics.It also potentially provide a reference to design effective HIV-1 immunogen.

Aberrant GATA2 Activation in Pediatric B-Cell Acute Lymphoblastic Leukemia.

GATA2 is a transcription factor that is critical for the generation and survival of hematopoietic stem cells (HSCs). It also plays an important role in the regulation of myeloid differentiation. Accordingly, GATA2 expression is restricted to HSCs and hematopoietic progenitors as well as early erythroid cells and megakaryocytic cells. Here we identified aberrant GATA2 expression in B-cell acute lymphoblastic leukemia (B-ALL) by analyzing transcriptome sequencing data obtained from St. Jude Cloud. Differentially expressed genes upon GATA2 activation showed significantly myeloid-like transcription signature. Further analysis identified several tumor-associated genes as targets of GATA2 activation including BAG3 and EPOR. In addition, the correlation between KMT2A-USP2 fusion and GATA2 activation not only indicates a potential trans-activating mechanism of GATA2 but also suggests that GATA2 is a target of KMT2A-USP2. Furthermore, by integrating whole-genome and transcriptome sequencing data, we showed that GATA2 is also cis activated. A somatic focal deletion located in the GATA2 neighborhood that disrupts the boundaries of topologically associating domains was identified in one B-ALL patient with GATA2 activation. These evidences support the hypothesis that GATA2 could be involved in leukemogenesis of B-ALL and can be transcriptionally activated through multiple mechanisms. The findings of aberrant activation of GATA2 and its molecular function extend our understanding of transcriptional factor dysregulation in B-ALL.

A somatic UBA2 variant preceded ETV6-RUNX1 in the concordant BCP-ALL of monozygotic twins

AbstractGenetic analysis of leukemic clones in monozygotic twins with concordant acute lymphoblastic leukemia (ALL) has proved a unique opportunity to gain insight into the molecular phylogenetics of leukemogenesis. Using whole-genome sequencing, we characterized constitutional and somatic single nucleotide variants/insertion-deletions (indels) and structural variants in a monozygotic twin pair with concordant ETV6-RUNX1+ B-cell precursor ALL (BCP-ALL). In addition, digital PCR (dPCR) was applied to evaluate the presence of and quantify selected somatic variants at birth, diagnosis, and remission. A shared somatic complex rearrangement involving chromosomes 11, 12, and 21 with identical fusion sequences in leukemias of both twins offered direct proof of a common clonal origin. The ETV6-RUNX1 fusion detected at diagnosis was found to originate from this complex rearrangement. A shared somatic frameshift deletion in UBA2 was also identified in diagnostic samples. In addition, each leukemia independently acquired analogous deletions of 3 genes recurrently targeted in BCP-ALLs (ETV6, ATF7IP, and RAG1/RAG2), providing evidence of a convergent clonal evolution only explained by a strong concurrent selective pressure. Quantification of the UBA2 deletion by dPCR surprisingly indicated it persisted in remission. This, for the first time to our knowledge, provided evidence of a UBA2 variant preceding the well-established initiating event ETV6-RUNX1. Further, we suggest the UBA2 deletion exerted a leukemia predisposing effect and that its essential role in Small Ubiquitin-like Modifier (SUMO) attachment (SUMOylation), regulating nearly all physiological and pathological cellular processes such as DNA-repair by nonhomologous end joining, may hold a mechanistic explanation for the predisposition.

Large Common Mitochondrial DNA Deletions Are Associated with a Mitochondrial SNP T14798C Near the 3′ Breakpoints

Introduction: Large somatic deletions of mitochondrial DNA (mtDNA) accumulate with aging in metabolically active tissues such as the brain. We have cataloged the breakpoints and frequencies of large mtDNA deletions in the human brain. Methods: We quantified 112 high-frequency mtDNA somatic deletions across four human brain regions with the Splice-Break2 pipeline. In addition, we utilized PLINK/Seq to test the association of mitochondrial genotypes with the abundance of these high-frequency mtDNA deletions. A conservative p value threshold of 5E−08 was used to find the significant loci. Results: One mtDNA SNP (T14798C) was significantly associated with mtDNA deletions in two brain regions, the dorsolateral prefrontal cortex (DLPFC) and the superior temporal gyrus. Since the DLPFC showed the most robust association between T14798C and two deletion breakpoints (7816–14807 and 5462–14807), this association was tested in the DLPFC of a replication sample and validated the first results. Incorporating the C allele at 14,798 bp increased the perfect/imperfect length of the repeat at the 3′ breakpoint of the two associated deletions. Conclusion: This is the first study to identify the association of mtDNA SNP with large mtDNA deletions in the human brain. The T14798C allele located in the MT-CYB gene is a common polymorphism that occurs in several mitochondrial haplogroups. We hypothesize that the T14798C association with two deletions occurs by extending the repeat length around the 3′ deletion breakpoints. This simple mechanism suggests that mtDNA SNPs can affect the mitochondrial genome structure, especially in brain where high levels of reactive oxygen species lead to deletion accumulation with aging.

Methodological approaches to the study of the determinants of somatic mitochondrial heteroplasmy in the elderly

The article substantiates the algorithm and methodology of the study aimed at search and analysis of the determinants of somatic mitochondrial heteroplasmy in the elderly people. Aging is considered as a process that has developed in the process of evolution, characterized by a decrease in all basic biological functions and accompanied by a gradual degradation of organs, tissues and systems of the body, a classification of the main hypotheses and theories of aging is given. The need to develop diagnostic criteria and markers to differentiate the concept of «healthy aging» from diseases of the elderly is noted. Separately, the authors analyze the role of mitochondria in the development of age-related changes associated with the development of neurodegeneration and muscular dystrophy. It has been noted that the development of senile changes is to some extent associated with the main function of mitochondria, namely the oxidation of nutrients and the production of ATP molecules, a universal energy source for most biological processes in cells. For the mitochondrial genome, a driving mechanism has been proposed to explain how mitochondrial somatic mutations can lead to certain aging phenotypes — neurodegeneration and muscular dystrophy. To establish the relationship between mitochondrial somatic deletions and age-related changes, an algorithm and methodology have been developed based on a comprehensive study of the determinants of somatic mitochondrial heteroplasmy in a cohort of elderly people. It is planned to analyze the dynamics of mitochondrial somatic mutations in muscle tissues (biopsies) for 450 elderly volunteers. The authors propose an original approach to the determination of mtDNA heteroplasmy and the detection of de-novo mutations, including the enrichment of mitochondrial fractions and the purification of mtDNA from muscle tissues, as well as a method for quantifying mtDNA copy number to normalize the amount of genomic material in the samples. The resulting material will be used to prepare libraries for sequencing on NGS platforms. Subsequently, the data after bioinformatic analysis will be compared with the results of neurological and psychological studies, including an assessment of the level of cognitive processes, the presence of asthenic disorders, and an assessment of the patient’s rehabilitation potential. It is assumed that this psychoneurological block will allow us to further distinguish at least three groups among volunteers, namely: persons with normal aging (healthy aging), patients with a weakening of mental and physical abilities associated with neurodegenerative or with vascular pathologies. In order to search for informative markers in the immune system that allow differentiating «healthy aging» from diseases of the elderly, it is proposed to perform a correlation analysis between the assessment of immune and cytokine status and the data of clinical and bioinformatic studies.