Distinct Set Of Mutations Research Articles

Joint inference of cell lineage and mitochondrial evolution from single-cell sequencing data.

Eukaryotic cells contain organelles called mitochondria that have their own genome. Most cells contain thousands of mitochondria which replicate, even in nondividing cells, by means of a relatively error-prone process resulting in somatic mutations in their genome. Because of the higher mutation rate compared to the nuclear genome, mitochondrial mutations have been used to track cellular lineage, particularly using single-cell sequencing that measures mitochondrial mutations in individual cells. However, existing methods to infer the cell lineage tree from mitochondrial mutations do not model "heteroplasmy," which is the presence of multiple mitochondrial clones with distinct sets of mutations in an individual cell. Single-cell sequencing data thus provide a mixture of the mitochondrial clones in individual cells, with the ancestral relationships between these clones described by a mitochondrial clone tree. While deconvolution of somatic mutations from a mixture of evolutionarily related genomes has been extensively studied in the context of bulk sequencing of cancer tumor samples, the problem of mitochondrial deconvolution has the additional constraint that the mitochondrial clone tree must be concordant with the cell lineage tree. We formalize the problem of inferring a concordant pair of a mitochondrial clone tree and a cell lineage tree from single-cell sequencing data as the Nested Perfect Phylogeny Mixture (NPPM) problem. We derive a combinatorial characterization of the solutions to the NPPM problem, and formulate an algorithm, MERLIN, to solve this problem exactly using a mixed integer linear program. We show on simulated data that MERLIN outperforms existing methods that do not model mitochondrial heteroplasmy nor the concordance between the mitochondrial clone tree and the cell lineage tree. We use MERLIN to analyze single-cell whole-genome sequencing data of 5220 cells of a gastric cancer cell line and show that MERLIN infers a more biologically plausible cell lineage tree and mitochondrial clone tree compared to existing methods. https://github.com/raphael-group/MERLIN.

Clonal Heterogeneity and Evolution during Treatment in High Hyperdiploid B-Cell Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, is caused by the accumulation of multiple genomic aberrations in developing lymphocytes. In children, high hyperdiploidy (HeH) is the most prevalent subtype of B-cell ALL (B-ALL), which is associated with a superior outcome. However, because of the high incidence of HeH B-ALL in children, this subtype is responsible for about 25% of all relapses. The prediction of relapse remains difficult, especially in these good risk subtypes. Recent studies indicate that ALL is a heterogeneous disease, with multiple leukemia clones at diagnosis, each having a distinct set of mutations. We hypothesize that these different subclones have a different sensitivity to treatment and that samples with more heterogeneity are more prone to relapse. We used single-cell sequencing to determine the clonal heterogeneity of HeH B-ALL at diagnosis and followed the clonal evolution during chemotherapy treatment. Methods: Bone marrow samples of 11 patients with HeH B-ALL were collected at diagnosis, during treatment (for 9 cases) and at relapse (for 1 case) (Table 1). Samples during treatment were enriched for leukemia cells using magnetic antibodies targeting cell membrane markers (CD10, CD19, CD34) with the MARS® gentle sorter. We performed targeted single-cell DNA sequencing with the Mission Bio Tapestri® Platform, using our custom amplicon panel of 414 amplicons across 39 commonly mutated genes. This method detected single nucleotide variants (SNVs) and small insertions/deletions (indels) based on targeted PCR amplification in about 4000 cells per sample. Results: We sequenced diagnostic samples from 11 HeH B-ALL patients. For 9 patients we also tested samples during treatment, either at day 15 of treatment (1 case) or at the end of induction (EOI) (8 cases), and for 3 cases an additional sample at the end of consolidation (EOC). Furthermore, for one patient, we analyzed samples of 3 subsequent relapses. In total, 117,148 single cells were processed, with an average of 4500 cells per sample. We detected multiple subclones in almost all diagnostic samples (9/11), ranging from 2 to 10 subclones per case. Most frequently mutated genes were KRAS, NRAS, PTPN11 and FLT3, as well as NSD2. Four cases had 2 or more different KRAS and/or NRAS subclones. Three cases had a subclone with a KRAS/ NRAS mutation co-occurring with a NSD2 mutation. For the patients with multiple subclones at diagnosis, we subsequently performed single-cell analysis on one or more follow-up samples, either during treatment or at relapse. In the samples taken at EOI, we detected residual leukemia cells in all 8 cases: multiple subclones were detected in 5 cases and only one subclone persisted in 3 cases. Residual leukemia cells were also detected in all samples collected at EOC, though in a lesser amount as compared to the previous timepoint sequenced. Interestingly, in the matched diagnosis-relapse case, we found that a minor subclone with a NSD2, PTPN11and KRAS mutation became the dominant clone at first relapse (Figure 1). Conclusions: Single-cell DNA sequencing reveals a strong genetic heterogeneity in HeH B-ALL based on a variety of mutations in KRAS, NRAS, PTPN11, FLT3 and NSD2. RAS mutations and FLT3 mutations are mutually exclusive in subclones, whereas NSD2 mutations and RAS mutations are frequently co-occurring in the same subclone. This cooperation is highlighted in our relapse patient, having a minor subclone with a KRAS and NSD2 mutation at diagnosis that became the largest subclone at relapse. Even though few leukemia cells persisted during treatment, we were able to genetically identify the residual subclones with a potentially higher therapy resistance.

The twin-beginnings of COVID-19 in Asia and Europe—one prevails quickly

In the spread of SARS-CoV-2, there have been multiple waves of replacement between strains, each of which having a distinct set of mutations. The first wave is a group of four mutations (C241T, C3037T, C14408T and A23403G [this being the amino acid change D614G]; all designated 0 to 1 below). This DG (D614G) group, fixed at the start of the pandemic, is the foundation of all subsequent waves of strains. Curiously, the DG group is absent in early Asian samples but present (and likely common) in Europe from the beginning. European data show that the high fitness of DG1111 requires the synergistic effect of all four mutations. However, the European strains would have had no time to evolve the four DG mutations (0 to 1), had they come directly from the early Asian DG0000 strain. Very likely, the European DG1111 strain had acquired the highly adaptive DG mutations in pre-pandemic Europe and had been spreading in parallel with the Asian strains. Two recent reports further support this twin-beginning interpretation. There was a period of two-way spread between Asia and Europe but, by May 2020, the European strains had supplanted the Asian strains globally. This large-scale replacement of one set of mutations for another has since been replayed many times as COVID-19 progresses.

The new UK SARS-CoV-2 variant and lockdown – causes and consequences

The new variant of concern (VOC), B.1.1.7, has a distinct set of mutations in nucleotides encoding the spike (S) protein on the surface of SARS-CoV-2. SARS-CoV-2 previously accumulated mutations at a much slower rate, of 1-2 per month; the sudden appearance of a large cluster of mutations was thought to be unusual. We now suspect that VOC may have arisen from immunosuppressed individuals who shed virus for longer periods. Epidemiological analyses estimate VOC to be more infectious; this is of most concern because these estimates were calculated during periods where many regions of the UK were in high social distancing restrictions. Therefore, the previous 'tiered' system implemented in the UK was ineffective at containing VOC. The most likely reason for this is that previous restrictions, no matter how strict, still allowed for gatherings in certain places. VOC also has implications for the national vaccination programme - a higher proportion of people will need to be vaccinated with a more infectious virus. Prolongation of the second dose of vaccines to increase vaccine uptake has understandably caused concern, but is based on sound immunological principles. There is now an urgent need to monitor the effect of new variants on vaccine efficacy - marking a new chapter in the global fight against COVID-19.

Novel Cellular Functions of Very Long Chain-Fatty Acids: Insight From ELOVL4 Mutations.

Elongation of Very Long chain fatty acids-4 (ELOVL4) protein is a member of the ELOVL family of fatty acid elongases that is collectively responsible for catalyzing formation of long chain fatty acids. ELOVL4 is the only family member that catalyzes production of Very Long Chain Saturated Fatty Acids (VLC-SFA) and Very Long Chain Polyunsaturated Fatty Acids (VLC-PUFA) with chain lengths ≥28 carbons. ELOVL4 and its VLC-SFA and VLC-PUFA products are emerging as important regulators of synaptic signaling and neuronal survival in the central nervous system (CNS). Distinct sets of mutations in ELOVL4 cause three different neurological diseases in humans. Heterozygous inheritance of one set of autosomal dominant ELOVL4 mutations that leads to truncation of the ELOVL4 protein causes Stargardt-like macular dystrophy (STGD3), an aggressive juvenile-onset retinal degeneration. Heterozygous inheritance of a different set of autosomal dominant ELOVL4 mutations that leads to a full-length protein with single amino acid substitutions causes spinocerebellar ataxia 34 (SCA34), a late-onset neurodegenerative disease characterized by gait ataxia and cerebellar atrophy. Homozygous inheritance of a different set of ELOVL4 mutations causes a more severe disease with infantile onset characterized by seizures, spasticity, intellectual disability, ichthyosis, and premature death. ELOVL4 is expressed widely in the CNS and is found primarily in neurons. ELOVL4 is expressed in cell-specific patterns within different regions of the CNS that are likely to be related to disease symptoms. In the retina, ELOVL4 is expressed exclusively in photoreceptors and produces VLC-PUFA that are incorporated into phosphatidylcholine and enriched in the light sensitive membrane disks of the photoreceptor outer segments. VLC-PUFA are enzymatically converted into “elovanoid” compounds that appear to provide paracrine signals that promote photoreceptor and neuronal survival. In the brain, the main ELOVL4 products are VLC-SFA that are incorporated into sphingolipids and enriched in synaptic vesicles, where they regulate kinetics of presynaptic neurotransmitter release. Understanding the function of ELOVL4 and its VLC-SFA and VLC-PUFA products will advance our understanding of basic mechanisms in neural signaling and has potential for developing novel therapies for seizure and neurodegenerative diseases.

Management of Patients With Adenocarcinoma or Squamous Cancer of the Esophagus

Esophageal cancer is characterized by early and frequent metastasis. Surgery is the primary treatment for early-stage disease, whereas patients with patients with locally advanced disease receive perioperative chemotherapy or chemoradiotherapy. Squamous cancers can be treated with primary chemoradiotherapy without surgery, depending on their response to therapy and patient tolerance for subsequent surgery. Chemotherapy with a fluorinated pyrimidine and a platinum agent, followed by later treatment with taxanes and irinotecan, provides some benefit. Agents that inhibit the erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2), or vascular endothelial growth factor, including trastuzumab, ramucirumab, and apatinib, increase response and survival times. Esophageal adenocarcinomas have mutations in tumor protein p53 and mutations that activate receptor-associated tyrosine kinase, vascular endothelial growth factor, and cell cycle pathways, whereas esophageal squamous tumors have a distinct set of mutations. Esophageal cancers develop systems to evade anti-tumor immune responses, but studies are needed to determine how immune checkpoint modification contributes to esophageal tumor development.

First Report of a Dominantly Inherited β-Thalassemia Caused by a Novel Elongated β-Globin Chain

A distinct set of mutations on the β-globin gene leads to dominantly inherited β-thalassemia (β-thal) that is associated with a disease phenotype in a single mutant copy. We described molecular and hematological characteristics of a novel elongated β-globin chain in combination with a known hemoglobin (Hb) variant (N-Baltimore or HBB: c.286A>G) in cis. The highly unstable Hb variant caused typical features of β-thal major (β-TM) or β-thal intermedia (β-TI) in two members of a family depending on their α-globin genotypes. The β mutant allele of the mother was transmitted in an autosomal dominant fashion to her daughter. They resemble severe forms of β-thal due to ineffective erythropoiesis. Taken together with previously published data, this result indicates that a dominant form of β-thal should be regarded as a phenotypic term of hemoglobinopathies caused by β chain variants that are highly unstable.

Mutations in epigenetic regulators are involved in acute lymphoblastic leukemia relapse following allogeneic hematopoietic stem cell transplantation.

Although steady improvements to chemotherapeutic treatments has helped cure 80% of childhood acute lymphoblastic leukemia (ALL) cases, chemotherapy has proven to be less effective in treating the majority of adult patients, leaving allogeneic hematopoietic stem cell transplantation (allo-HSCT) as the primary adult treatment option. Nevertheless relapse are the leading cause of death following allo-HSCT. The genetic pathogenesis of relapse following allo-HSCT in Philadelphia chromosome- negative ALL (Ph− ALL) remains unexplored. We performed longitudinal whole-exome sequencing analysis in three adult patients with Ph− B-cell ALL (Ph− B-ALL) on samples collected from diagnosis to relapse after allo-HSCT. Based on these data, we performed target gene sequencing on 23 selected genes in 58 adult patients undergoing allo-HSCT with Ph− B-ALL. Our results revealed a significant enrichment of mutations in epigenetic regulators from relapsed samples, with recurrent somatic mutations in SETD2, CREBBP, KDM6A and NR3C1. The relapsed samples were also enriched in signaling factor mutations, including KRAS, PTPN21, MYC and USP54. Furthermore, we are the first to reveal the clonal evolution patterns during leukemia relapse after allo-HSCT. Cells present in relapsed specimens were genetically related to the diagnosed tumor, these cells therefore arose from either an existing subclone that was not eradicated by allo-HSCT therapy, or from the same progenitor that acquired new mutations. In some cases, however, it is possible that leukemia recurrence following allo-HSCT could result from a secondary malignancy with a distinct set of mutations. We identified novel genetic causes of leukemia relapse after allo-HSCT using the largest generated data set to date from adult patients with Ph− B-ALL.

NF1 Mutations Are Common in Desmoplastic Melanoma.

Desmoplastic melanoma (DM) is a rare variant of melanoma with distinct clinical, histopathologic, and immunohistochemical features. Clinically, DM differs from conventional melanoma by a higher propensity for local recurrence and less frequent metastatic spread to regional lymph nodes. In its pure form, DM has a distinct appearance displaying a low density of fusiform melanocytes in a collagen-rich matrix. Whereas a number of mutations have been identified in primary melanoma, including BRAF, NRAS, GNAQ, GNA11, and KIT, and the occurrence of these mutations has been found to correlate to some extent with the histopathologic features, anatomic site, and/or mode of sun exposure, no distinct set of mutations has so far been reported for DM. To study the potential association of neurofibromin (NF1) mutations with DM, we examined 15 desmoplastic and 20 non-DMs by next-generation sequencing. Mutations of the NF1 gene were found in 14 of 15 (93%) DMs and 4 of 20 (20%) non-DMs. The high frequency of NF1 mutations in DMs suggests an important role for NF1 in the biology of this type of melanoma.

Identification of somatic and germline mutations using whole exome sequencing of congenital acute lymphoblastic leukemia

BackgroundAcute lymphoblastic leukemia (ALL) diagnosed within the first month of life is classified as congenital ALL and has a significantly worse outcome than ALL diagnosed in older children. This suggests that congenital ALL is a biologically different disease, and thus may be caused by a distinct set of mutations. To understand the somatic and germline mutations contributing to congenital ALL, the protein-coding regions in the genome were captured and whole-exome sequencing was employed for the identification of single-nucleotide variants and small insertion and deletions in the germlines as well as the primary tumors of four patients with congenital ALL.MethodsExome sequencing was performed on Illumina GAIIx or HiSeq 2000 (Illumina, San Diego, California). Reads were aligned to the human reference genome and the Genome Analysis Toolkit was used for variant calling. An in-house developed Ensembl-based variant annotator was used to richly annotate each variant.ResultsThere were 1–3 somatic, protein-damaging mutations per ALL, including a novel mutation in Sonic Hedgehog. Additionally, there were many germline mutations in genes known to be associated with cancer predisposition, as well as genes involved in DNA repair.ConclusionThis study is the first to comprehensively characterize the germline and somatic mutational profile of all protein-coding genes patients with congenital ALL. These findings identify potentially important therapeutic targets, as well as insight into possible cancer predisposition genes.

Analysis of Tumor Metabolism Reveals Mitochondrial Glucose Oxidation in Genetically Diverse Human Glioblastomas in the Mouse Brain In Vivo

Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused (13)C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.

Darunavir: an effective protease inhibitor for HIV-infected patients

Darunavir is a new-generation nonpeptidic HIV protease inhibitor (PI), used with low doses of ritonavir for pharmacologic enhancement (boosting). It has demonstrated potent activity against multidrug-resistant HIV, with a robust resistance profile and a distinct set of mutations. In heavily treatment-experienced patients with HIV infection, darunavir administered twice daily with ritonavir has shown higher rates of efficacy than the control PI. In less treatment-experienced patients, boosted darunavir was noninferior to boosted lopinavir. In treatment-naive patients, boosted darunavir administered once daily was noninferior to boosted lopinavir, and showed higher virologic and immunological response rates in patients with high baseline viral load and low baseline CD4+ cell counts. Monotherapy with boosted darunavir is an acceptable option in some specific conditions. Boosted darunavir was generally well tolerated, with lower incidence of diarrhea and a more favorable lipid profile than boosted lopinavir in treatment-naive patients.

Identification of Separate Structural Features That Affect Rate and Cation Concentration Dependence of Self-Cleavage by the Neurospora VS Ribozyme

The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg(2+) requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg(2+) requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li(+) instead of Mg(2+). Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA.

Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene.

The human mast cell line (HMC)-1 cell line is growth-factor independent because of a constitutive activity of the receptor tyrosine kinase Kit. Such deregulated Kit activity has also been suggested causative in gastrointestinal stromal tumours (GISTs) and mastocytosis. HMC-1 is the only established continuously growing human mast cell line and has therefore been widely employed for in vitro studies of human mast cell biology. In this paper we describe two sublines of HMC-1, named HMC-1(560 ) and HMC-1(560,816 ), with different phenotypes and designated by the locations of specific mutations in the c-kit proto-oncogene. Activating mutations in the Kit receptor were characterized using the pyrosequencing trade mark method. Both sublines have a heterozygous T to G mutation at codon 560 in the juxtamembrane region of the c-kit gene causing an amino acid substitution of Gly-560 for Val. In contrast, only HMC-1(560,816) cells have the c-kitV816 mutation found in mast cell neoplasms causing an Asp-->Val substitution in the intracellular kinase domain. Kit was constitutively phosphorylated on tyrosine residues and associated with phosphatidylinositol 3'-kinase (PI 3-kinase) in both variants of HMC-1, but this did not lead to a constitutive phosphorylation of Akt or extracellular regulated protein kinase (ERK), which are signalling molecules normally activated by the interaction of stem cell factor (SCF) with Kit. The documentation and characterization of two sublines of HMC-1 cells provides both information on the biological consequences of mutations in Kit and recognition of the availability of what in reality are two distinct cultured human mast cell lines.