Publications by authors named "Malik Alawi"

87 Publications

Diagnostic potential of extracellular vesicles in meningioma patients.

Neuro Oncol 2022 May 12. Epub 2022 May 12.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Extracellular vesicles (EVs) play an important role in cell-cell communication, and tumor-derived EVs circulating in patient blood can serve as biomarkers. Here, we investigated the potential role of plasma EVs in meningioma patients for tumor detection and determined whether EVs secreted by meningioma cells reflect epigenetic, genomic and proteomic alterations of original tumors.

Methods: EV concentrations were quantified in patient plasma (n = 46). Short-term meningioma cultures were established (n = 26) and secreted EVs were isolated. Methylation and copy number profiling was performed using 850k arrays, and mutations were identified by targeted gene panel sequencing. Differential quantitative mass spectrometry was employed for proteomic analysis.

Results: Levels of circulating EVs were elevated in meningioma patients compared to healthy individuals, and the plasma EV concentration correlated with malignancy grade and extent of peritumoral edema. Postoperatively, EV counts dropped to normal levels, and the magnitude of the postoperative decrease was associated with extent of tumor resection. Methylation profiling of EV-DNA allowed correct tumor classification as meningioma in all investigated cases, and accurate methylation subclass assignment in almost all cases. Copy number variations present in tumors, as well as tumor-specific mutations were faithfully reflected in meningioma EV-DNA. Proteomic EV profiling did not permit original tumor identification but revealed tumor-associated proteins that could potentially be utilized to enrich meningioma EVs from biofluids.

Conclusions: Elevated EV levels in meningioma patient plasma could aid in tumor diagnosis and assessment of treatment response. Meningioma EV-DNA mirrors genetic and epigenetic tumor alterations and facilitates molecular tumor classification.
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http://dx.doi.org/10.1093/neuonc/noac127DOI Listing
May 2022

Cardiofacioneurodevelopmental syndrome: Report of a novel patient and expansion of the phenotype.

Am J Med Genet A 2022 Apr 22. Epub 2022 Apr 22.

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

The cardiofacioneurodevelopmental syndrome (CFNDS) is characterized by craniofacial anomalies including bilateral cleft lip and palate, cardiac, skeletal, and neurodevelopmental features and additional variable manifestations. Whole-exome sequencing revealed homozygous loss-of-function variants in CCDC32 (alternative name: C15orf57) in both previously described patients. ccdc32 deletion in zebrafish suggests a ciliary contribution to the pathomechanism. We report a 9-year-old female patient with CFNDS caused by a homozygous 32,583-bp deletion affecting CCDC32. Independent of the affected CCDC32 transcript variant this deletion likely leads to loss of the encoded protein. The patient had intellectual disability, marked hypertelorism, bilateral cleft lip and palate, and short stature. She had bilateral conductive hearing loss, small hands and feet, and finger abnormalities. Brain imaging disclosed hypoplastic corpus callosum. We describe a core phenotype comprising developmental delay and bilateral cleft lip and palate in the three individuals with CFNDS. Variable abnormalities of the face, brain, heart, fingers, and toes and postnatal growth retardation or microcephaly can be present. Possible involvement of the uncharacterized CCDC32 protein in the adapter protein 2 (AP2) complex regulating clathrin-mediated endocytosis has been reported. Cleft palate and cardiac defects observed in mice deficient of different AP2 subunits support a CCDC32 function in the AP2 complex.
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http://dx.doi.org/10.1002/ajmg.a.62762DOI Listing
April 2022

Comprehensive profiling of myxopapillary ependymomas identifies a distinct molecular subtype with relapsing disease.

Neuro Oncol 2022 Apr 5. Epub 2022 Apr 5.

Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients' clinical course are unknown.

Methods: We assembled a cohort of 185 tumors classified as MPE based on DNA methylation. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPEB, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors.

Results: MPE-A occurred at a median age of 27 years and were enriched with tumors demonstrating papillary morphology and MGMT promoter hypermethylation. Half of these tumors could not be totally resected, and 85% relapsed within 10 years. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. MPE-B occurred at a median age of 45 years and included many tumors with a histological diagnosis of WHO grade II and tanycytic morphology. Patients within this subtype had a significantly better outcome with a relapse rate of 33% in 10 years (p=3.4e-06).

Conclusions: We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.
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http://dx.doi.org/10.1093/neuonc/noac088DOI Listing
April 2022

Novel biallelic variants expand the SLC5A6-related phenotypic spectrum.

Eur J Hum Genet 2022 04 11;30(4):439-449. Epub 2022 Jan 11.

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246, Hamburg, Germany.

The sodium (Na):multivitamin transporter (SMVT), encoded by SLC5A6, belongs to the sodium:solute symporter family and is required for the Na-dependent uptake of biotin (vitamin B7), pantothenic acid (vitamin B5), the vitamin-like substance α-lipoic acid, and iodide. Compound heterozygous SLC5A6 variants have been reported in individuals with variable multisystemic disorder, including failure to thrive, developmental delay, seizures, cerebral palsy, brain atrophy, gastrointestinal problems, immunodeficiency, and/or osteopenia. We expand the phenotypic spectrum associated with biallelic SLC5A6 variants affecting function by reporting five individuals from three families with motor neuropathies. We identified the homozygous variant c.1285 A > G [p.(Ser429Gly)] in three affected siblings and a simplex patient and the maternally inherited c.280 C > T [p.(Arg94*)] variant and the paternally inherited c.485 A > G [p.(Tyr162Cys)] variant in the simplex patient of the third family. Both missense variants were predicted to affect function by in silico tools. 3D homology modeling of the human SMVT revealed 13 transmembrane helices (TMs) and Tyr162 and Ser429 to be located at the cytoplasmic facing region of TM4 and within TM11, respectively. The SLC5A6 missense variants p.(Tyr162Cys) and p.(Ser429Gly) did not affect plasma membrane localization of the ectopically expressed multivitamin transporter suggesting reduced but not abolished function, such as lower catalytic activity. Targeted therapeutic intervention yielded clinical improvement in four of the five patients. Early molecular diagnosis by exome sequencing is essential for timely replacement therapy in affected individuals.
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http://dx.doi.org/10.1038/s41431-021-01033-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8747999PMC
April 2022

Transcriptome Analysis in Vulvar Squamous Cell Cancer.

Cancers (Basel) 2021 Dec 19;13(24). Epub 2021 Dec 19.

Department of Gynecology and Gynecologic Oncology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

To date, therapeutic strategies in vulvar squamous cell carcinoma (VSCC) are lacking molecular pathological information and targeted therapy hasn't been approved in the treatment of VSCC, yet. Two etiological pathways are widely accepted: HPV induced vs. HPV independent, associated with chronic skin disease, often harboring mutations (mut). The aim of this analysis was to analyze the RNA expression patterns for subtype stratification on VSCC samples that can be integrated into the previously performed whole exome sequencing data for the detection of prognostic markers and potential therapeutic targets. We performed multiplex gene expression analysis (NanoString) with 770 genes in 24 prior next generation sequenced samples. An integrative data analysis was performed. Here, 98 genes were differentially expressed in TP53mut vs. HPV+ VSCC, in the TP53mut cohort, where 56 genes were upregulated and 42 were downregulated in comparison to the HPV+ tumors. Aberrant expression was primarily observed in cell cycle regulation, especially in HPV+ disease. Within the TP53mut group, a distinct cluster was identified that was correlated to a significantly worse overall survival ( = 0.017). The RNA expression profiles showed distinct patterns with regard to the known VSCC subtypes and could potentially enable further subclassification in the TP53mut groups.
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http://dx.doi.org/10.3390/cancers13246372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8699756PMC
December 2021

Biallelic FRA10AC1 variants cause a neurodevelopmental disorder with growth retardation.

Brain 2021 Oct 25. Epub 2021 Oct 25.

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

The major spliceosome mediates pre-mRNA splicing by recognizing the highly conserved sequences at the 5' and 3' splice sites and the branch point. More than 150 proteins participate in the splicing process and are organized in the spliceosomal A, B, and C complexes. FRA10AC1 is a peripheral protein of the spliceosomal C complex and its ortholog in the green alga facilitates recognition or interaction with splice sites. We identified biallelic pathogenic variants in FRA10AC1 in five individuals from three consanguineous families. The two unrelated patients 1 and 2 with loss-of-function variants showed developmental delay, intellectual disability, and no speech, while three siblings with the c.494_496delAAG (p.Glu165del) variant had borderline to mild intellectual disability. All patients had microcephaly, hypoplasia or agenesis of the corpus callosum, growth retardation, and craniofacial dysmorphism. FRA10AC1 transcripts and proteins were drastically reduced or absent in fibroblasts of patients 1 and 2. In a heterologous expression system, the p. Glu165del variant impacts intrinsic stability of FRA10AC1 but does not affect its nuclear localization. By co-immunoprecipitation, we found ectopically expressed HA-FRA10AC1 in complex with endogenous DGCR14, another component of the spliceosomal C complex, while the splice factors CHERP, NKAP, RED, and SF3B2 could not be co-immunoprecipitated. Using an in vitro splicing reporter assay, we did not obtain evidence for FRA10AC1 deficiency to suppress missplicing events caused by mutations in the highly conserved dinucleotides of 5' and 3' splice sites in an in vitro splicing assay in patient-derived fibroblasts. Our data highlight the importance of specific peripheral spliceosomal C complex proteins for neurodevelopment. It remains possible that FRA10AC1 may have other and/or additional cellular functions, such as coupling of transcription and splicing reactions.
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http://dx.doi.org/10.1093/brain/awab403DOI Listing
October 2021

Deep (Meta)genomics and (Meta)transcriptome Analyses of Fungal and Bacteria Consortia From Aircraft Tanks and Kerosene Identify Key Genes in Fuel and Tank Corrosion.

Front Microbiol 2021 1;12:722259. Epub 2021 Oct 1.

Department of Microbiology and Biotechnology, Institute of Plant Science and Microbiology, University of Hamburg, Hamburg, Germany.

Microbial contamination of fuels, associated with a wide variety of bacteria and fungi, leads to decreased product quality and can compromise equipment performance by biofouling or microbiologically influenced corrosion. Detection and quantification of microorganisms are critical in monitoring fuel systems for an early detection of microbial contaminations. To address these challenges, we have analyzed six metagenomes, one transcriptome, and more than 1,200 fluid and swab samples taken from fuel tanks or kerosene. Our deep metagenome sequencing and binning approaches in combination with RNA-seq data and qPCR methods implied a metabolic symbiosis between fungi and bacteria. The most abundant bacteria were affiliated with α-, β-, and γ-Proteobacteria and the filamentous fungi . We identified a high number of genes, which are related to kerosene degradation and biofilm formation. Surprisingly, a large number of genes coded enzymes involved in polymer degradation and potential bio-corrosion processes. Thereby, the transcriptionally most active microorganisms were affiliated with the genera . Many not yet cultured bacteria and fungi appeared to contribute to the biofilm transcriptional activities. The largest numbers of transcripts were observed for dehydrogenase, oxygenase, and exopolysaccharide production, attachment and pili/flagella-associated proteins, efflux pumps, and secretion systems as well as lipase and esterase activity.
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http://dx.doi.org/10.3389/fmicb.2021.722259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525681PMC
October 2021

Vertically transferred maternal immune cells promote neonatal immunity against early life infections.

Nat Commun 2021 08 4;12(1):4706. Epub 2021 Aug 4.

Division of Experimental Feto-Maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg, Hamburg, Germany.

During mammalian pregnancy, immune cells are vertically transferred from mother to fetus. The functional role of these maternal microchimeric cells (MMc) in the offspring is mostly unknown. Here we show a mouse model in which MMc numbers are either normal or low, which enables functional assessment of MMc. We report a functional role of MMc in promoting fetal immune development. MMc induces preferential differentiation of hematopoietic stem cells in fetal bone marrow towards monocytes within the myeloid compartment. Neonatal mice with higher numbers of MMc and monocytes show enhanced resilience against cytomegalovirus infection. Similarly, higher numbers of MMc in human cord blood are linked to a lower number of respiratory infections during the first year of life. Our data highlight the importance of MMc in promoting fetal immune development, potentially averting the threats caused by early life exposure to pathogens.
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http://dx.doi.org/10.1038/s41467-021-24719-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8338998PMC
August 2021

Hypoimmune induced pluripotent stem cell-derived cell therapeutics treat cardiovascular and pulmonary diseases in immunocompetent allogeneic mice.

Proc Natl Acad Sci U S A 2021 07;118(28)

Division of Cardiothoracic Surgery, Department of Surgery, Transplant and Stem Cell Immunobiology Laboratory, University of California, San Francisco, CA 94143;

The emerging field of regenerative cell therapy is still limited by the few cell types that can reliably be differentiated from pluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.
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http://dx.doi.org/10.1073/pnas.2022091118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285900PMC
July 2021

Biallelic variants in VPS50 cause a neurodevelopmental disorder with neonatal cholestasis.

Brain 2021 11;144(10):3036-3049

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes are membrane-tethering heterotetramers located at the trans-Golgi network and recycling endosomes, respectively. GARP and EARP share the three subunits VPS51, VPS52 and VPS53, while VPS50 is unique to EARP and VPS54 to GARP. Retrograde transport of endosomal cargos to the trans-Golgi network is mediated by GARP and endocytic recycling by EARP. Here we report two unrelated individuals with homozygous variants in VPS50, a splice variant (c.1978-1G>T) and an in-frame deletion (p.Thr608del). Both patients had severe developmental delay, postnatal microcephaly, corpus callosum hypoplasia, seizures and irritability, transient neonatal cholestasis and failure to thrive. Light and transmission electron microscopy of liver from one revealed the absence of gamma-glutamyltransferase at bile canaliculi, with mislocalization to basolateral membranes and abnormal tight junctions. Using patient-derived fibroblasts, we identified reduced VPS50 protein accompanied by reduced levels of VPS52 and VPS53. While the transferrin receptor internalization rate was normal in cells of both patients, recycling of the receptor to the plasma membrane was significantly delayed. These data underscore the importance of VPS50 and/or the EARP complex in endocytic recycling and suggest an additional function in establishing cell polarity and trafficking between basolateral and apical membranes in hepatocytes. Individuals with biallelic hypomorphic variants in VPS50, VPS51 or VPS53 show an overarching neurodegenerative disorder with severe developmental delay, intellectual disability, microcephaly, early-onset epilepsy and variable atrophy of the cerebellum, cerebrum and/or brainstem. The term 'GARP/EARP deficiency' designates disorders in such individuals.
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http://dx.doi.org/10.1093/brain/awab206DOI Listing
November 2021

Distinct clonal lineages and within-host diversification shape invasive Staphylococcus epidermidis populations.

PLoS Pathog 2021 02 5;17(2):e1009304. Epub 2021 Feb 5.

Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

S. epidermidis is a substantial component of the human skin microbiota, but also one of the major causes of nosocomial infection in the context of implanted medical devices. We here aimed to advance the understanding of S. epidermidis genotypes and phenotypes conducive to infection establishment. Furthermore, we investigate the adaptation of individual clonal lines to the infection lifestyle based on the detailed analysis of individual S. epidermidis populations of 23 patients suffering from prosthetic joint infection. Analysis of invasive and colonizing S. epidermidis provided evidence that invasive S. epidermidis are characterized by infection-supporting phenotypes (e.g. increased biofilm formation, growth in nutrient poor media and antibiotic resistance), as well as specific genetic traits. The discriminating gene loci were almost exclusively assigned to the mobilome. Here, in addition to IS256 and SCCmec, chromosomally integrated phages was identified for the first time. These phenotypic and genotypic features were more likely present in isolates belonging to sequence type (ST) 2. By comparing seven patient-matched nasal and invasive S. epidermidis isolates belonging to identical genetic lineages, infection-associated phenotypic and genotypic changes were documented. Besides increased biofilm production, the invasive isolates were characterized by better growth in nutrient-poor media and reduced hemolysis. By examining several colonies grown in parallel from each infection, evidence for genetic within-host population heterogeneity was obtained. Importantly, subpopulations carrying IS insertions in agrC, mutations in the acetate kinase (AckA) and deletions in the SCCmec element emerged in several infections. In summary, these results shed light on the multifactorial processes of infection adaptation and demonstrate how S. epidermidis is able to flexibly repurpose and edit factors important for colonization to facilitate survival in hostile infection environments.
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http://dx.doi.org/10.1371/journal.ppat.1009304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891712PMC
February 2021

The SIRPα-CD47 immune checkpoint in NK cells.

J Exp Med 2021 03;218(3)

Department of Surgery, Division of Cardiothoracic Surgery, Transplant and Stem Cell Immunobiology Lab, University of California, San Francisco, San Francisco, CA.

Here we report on the existence and functionality of the immune checkpoint signal regulatory protein α (SIRPα) in NK cells and describe how it can be modulated for cell therapy. NK cell SIRPα is up-regulated upon IL-2 stimulation, interacts with target cell CD47 in a threshold-dependent manner, and counters other stimulatory signals, including IL-2, CD16, or NKG2D. Elevated expression of CD47 protected K562 tumor cells and mouse and human MHC class I-deficient target cells against SIRPα+ primary NK cells, but not against SIRPα- NKL or NK92 cells. SIRPα deficiency or antibody blockade increased the killing capacity of NK cells. Overexpression of rhesus monkey CD47 in human MHC-deficient cells prevented cytotoxicity by rhesus NK cells in a xenogeneic setting. The SIRPα-CD47 axis was found to be highly species specific. Together, the results demonstrate that disruption of the SIRPα-CD47 immune checkpoint may augment NK cell antitumor responses and that elevated expression of CD47 may prevent NK cell-mediated killing of allogeneic and xenogeneic tissues.
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http://dx.doi.org/10.1084/jem.20200839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802363PMC
March 2021

Bi-allelic Pathogenic Variants in HS2ST1 Cause a Syndrome Characterized by Developmental Delay and Corpus Callosum, Skeletal, and Renal Abnormalities.

Am J Hum Genet 2020 12 6;107(6):1044-1061. Epub 2020 Nov 6.

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. Electronic address:

Heparan sulfate belongs to the group of glycosaminoglycans (GAGs), highly sulfated linear polysaccharides. Heparan sulfate 2-O-sulfotransferase 1 (HS2ST1) is one of several specialized enzymes required for heparan sulfate synthesis and catalyzes the transfer of the sulfate groups to the sugar moiety of heparan sulfate. We report bi-allelic pathogenic variants in HS2ST1 in four individuals from three unrelated families. Affected individuals showed facial dysmorphism with coarse face, upslanted palpebral fissures, broad nasal tip, and wide mouth, developmental delay and/or intellectual disability, corpus callosum agenesis or hypoplasia, flexion contractures, brachydactyly of hands and feet with broad fingertips and toes, and uni- or bilateral renal agenesis in three individuals. HS2ST1 variants cause a reduction in HS2ST1 mRNA and decreased or absent heparan sulfate 2-O-sulfotransferase 1 in two of three fibroblast cell lines derived from affected individuals. The heparan sulfate synthesized by the individual 1 cell line lacks 2-O-sulfated domains but had an increase in N- and 6-O-sulfated domains demonstrating functional impairment of the HS2ST1. As heparan sulfate modulates FGF-mediated signaling, we found a significantly decreased activation of the MAP kinases ERK1/2 in FGF-2-stimulated cell lines of affected individuals that could be restored by addition of heparin, a GAG similar to heparan sulfate. Focal adhesions in FGF-2-stimulated fibroblasts of affected individuals concentrated at the cell periphery. Our data demonstrate that a heparan sulfate synthesis deficit causes a recognizable syndrome and emphasize a role for 2-O-sulfated heparan sulfate in human neuronal, skeletal, and renal development.
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http://dx.doi.org/10.1016/j.ajhg.2020.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820632PMC
December 2020

Differential regulation of extracellular matrix proteins in three recurrent liver metastases of a single patient with colorectal cancer.

Clin Exp Metastasis 2020 12 24;37(6):649-656. Epub 2020 Oct 24.

Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246, Hamburg, Germany.

Colorectal cancer (CRC) patients suffer from the second highest mortality among all cancer entities. In half of all CRC patients, colorectal cancer liver metastases (CRLM) can be observed. Metastatic colorectal cancer is associated with poor overall survival and limited treatment options. Even after successful surgical resection of the primary tumor, metachronous liver metastases occur in one out of eight cases. The only available curative intended treatment is hepatic resection, but metachronous CRLM frequently recur after approximately 1 year. In this study, we performed a proteome analysis of three recurrent liver metastases of a single CRC patient by mass spectrometry. Despite surgical resection of the primary CRC and adjuvant chemotherapy plus cetuximab treatment, the patient developed three metachronous CRLM which occurred consecutively after 9, 21 and 31 months. We identified a set of 1132 proteins expressed in the three metachronous CRLM, of which 481 were differentially regulated, including 81 proteins that were associated with the extracellular matrix (ECM). 56 ECM associated proteins were identified as upregulated in the third metastasis, 26 (46%) of which were previously described as negative prognostic markers in CRC, including tenascin C, nidogen 1, fibulin 1 and vitronectin. These data may reflect an ascending trend of malignancy from the first to the third metachronous colorectal cancer liver metastasis. Additionally, the results indicate different ECM phenotypes for recurrent metachronous metastasis, associated with different grades of malignancy and highlights the importance of individual analysis of molecular features in different, consecutive metastatic events in a single patient.
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http://dx.doi.org/10.1007/s10585-020-10058-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666585PMC
December 2020

High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms.

PLoS Pathog 2020 08 24;16(8):e1008562. Epub 2020 Aug 24.

Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.
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http://dx.doi.org/10.1371/journal.ppat.1008562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470373PMC
August 2020

Biallelic MADD variants cause a phenotypic spectrum ranging from developmental delay to a multisystem disorder.

Brain 2020 08;143(8):2437-2453

Department of Medical Genetics, University of Antwerp, Antwerp, Belgium.

In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands.
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http://dx.doi.org/10.1093/brain/awaa204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447524PMC
August 2020

Biallelic loss-of-function variants in TBC1D2B cause a neurodevelopmental disorder with seizures and gingival overgrowth.

Hum Mutat 2020 09 15;41(9):1645-1661. Epub 2020 Jul 15.

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

The family of Tre2-Bub2-Cdc16 (TBC)-domain containing GTPase activating proteins (RABGAPs) is not only known as key regulatorof RAB GTPase activity but also has GAP-independent functions. Rab GTPases are implicated in membrane trafficking pathways, such as vesicular trafficking. We report biallelic loss-of-function variants in TBC1D2B, encoding a member of the TBC/RABGAP family with yet unknown function, as the underlying cause of cognitive impairment, seizures, and/or gingival overgrowth in three individuals from unrelated families. TBC1D2B messenger RNA amount was drastically reduced, and the protein was absent in fibroblasts of two patients. In immunofluorescence analysis, ectopically expressed TBC1D2B colocalized with vesicles positive for RAB5, a small GTPase orchestrating early endocytic vesicle trafficking. In two independent TBC1D2B CRISPR/Cas9 knockout HeLa cell lines that serve as cellular model of TBC1D2B deficiency, epidermal growth factor internalization was significantly reduced compared with the parental HeLa cell line suggesting a role of TBC1D2B in early endocytosis. Serum deprivation of TBC1D2B-deficient HeLa cell lines caused a decrease in cell viability and an increase in apoptosis. Our data reveal that loss of TBC1D2B causes a neurodevelopmental disorder with gingival overgrowth, possibly by deficits in vesicle trafficking and/or cell survival.
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http://dx.doi.org/10.1002/humu.24071DOI Listing
September 2020

Glioma escape signature and clonal development under immune pressure.

J Clin Invest 2020 10;130(10):5257-5271

Department of Neurosurgery and.

Immunotherapeutic strategies are increasingly important in neuro-oncology, and the elucidation of escape mechanisms that lead to treatment resistance is crucial. We investigated the impact of immune pressure on the clonal dynamics and immune escape signature by comparing glioma growth in immunocompetent versus immunodeficient mice. Glioma-bearing WT and Pd-1-/- mice survived significantly longer than immunodeficient Pfp-/- Rag2-/- mice. While tumors in Pfp-/- Rag2-/- mice were highly polyclonal, immunoedited tumors in WT and Pd-1-/- mice displayed reduced clonality with emergence of immune escape clones. Tumor cells in WT mice were distinguished by an IFN-γ-mediated response signature with upregulation of genes involved in immunosuppression. Tumor-infiltrating stromal cells, which include macrophages/microglia, contributed even more strongly to the immunosuppressive signature than the actual tumor cells. The identified murine immune escape signature was reflected in human patients and correlated with poor survival. In conclusion, immune pressure profoundly shapes the clonal composition and gene regulation in malignant gliomas.
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http://dx.doi.org/10.1172/JCI138760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524465PMC
October 2020

Genomic characterization of vulvar squamous cell carcinoma.

Gynecol Oncol 2020 09 24;158(3):547-554. Epub 2020 Jun 24.

Department of Gynaecology and Gynaecologic Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Despite increasing incidence, vulvar squamous cell carcinoma (VSCC) is still a rare disease. Until now, two etiological pathways have been described: a high-risk human papillomavirus (HPV)-dependent route and an HPV-independent pathway often associated with lichen sclerosus. To date, therapeutic strategies in VSCC are not influenced by molecular pathological information and therapeutic options for advanced or recurrent disease are limited.

Methods: Whole exome sequencing of DNA, isolated from 34 VSCC samples and matched normal tissue for each individual was performed on an Illumina HiSeq4000. Short variant discovery was carried out using BWA mem and FreeBayes. Variants were annotated using ANNOVAR.

Results: FIGO stages were: IB (n = 7), II (n = 11), III (n = 8), and IVA (n = 3), (n = 5 unknown). TP53 missense mutations were most commonly detected with 56% (19/34). 12/34 (35.3%) samples were HPV positive (all HPV16), HPV positivity and TP53 mutations were mutually exclusive (p < .0001). Additionally, we observed mutations in known cancer relevant genes, like NBPF1 (n = 7), MACF1 (n = 5), SYNE2 (n = 5), DOCK2 (n = 4), KMT2D (n = 4), MAP2 (n = 4), NACA (n = 4), PIK3CA (n = 4), SYNE1 (n = 4), FBWX7 (n = 3), MSH6 (n = 3), NSD1 (n = 3), POLE (n = 3), TSC2, (n = 3) and CDKN2A (n = 2), but at considerably lower frequencies. For the total cohort 1848 cancer related mutations were detected (median of 54.4 per sample).

Conclusions: The key mutation in HPV negative vulvar carcinoma affects TP53. While a multitude of cancer related mutations was detected in various samples, only few mutations recur and/or affect concurrent signaling pathways.
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http://dx.doi.org/10.1016/j.ygyno.2020.06.482DOI Listing
September 2020

Draft Genome Sequence of the Green Alga Scenedesmus acuminatus SAG 38.81.

Microbiol Resour Announc 2020 Jun 11;9(24). Epub 2020 Jun 11.

Universität Hamburg, Institute of Plant Science and Microbiology, Department of Microbiology and Biotechnology, Hamburg, Germany

, also known as , is a promising green microalga for sustainable production of microalga products, including valuable compounds such as astaxanthin, β-carotene, and lutein, polysaccharides such as β-glucan, and polyunsaturated fatty acids. Here, we report the draft whole-genome sequence of SAG 38.81.
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http://dx.doi.org/10.1128/MRA.01278-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291106PMC
June 2020

Exome sequencing in 38 patients with intracranial aneurysms and subarachnoid hemorrhage.

J Neurol 2020 Sep 4;267(9):2533-2545. Epub 2020 May 4.

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246, Hamburg, Germany.

Objective: Genetic risk factors for unruptured intracranial aneurysms (UIA) and aneurysmal subarachnoid hemorrhage (aSAH) are poorly understood. We aimed to verify recently reported risk genes and to identify novel sequence variants involved in the etiology of UIA/aSAH.

Methods: We performed exome sequencing (ES) in 35 unrelated individuals and 3 family members, each with a history of UIA and/or aSAH. We searched for sequence variants with minor allele frequency (MAF) ≤ 5% in the reported risk genes ADAMTS15, ANGPTL6, ARHGEF17, LOXL2, PCNT, RNF213, THSD1 and TMEM132B. To identify novel putative risk genes we looked for unknown (MAF = 0) variants shared by the three relatives.

Results: We identified 20 variants with MAF ≤ 5% in 18 individuals: 9 variants in PCNT (9 patients), 4 in RNF213 (3 patients), 3 in THSD1 (6 patients), 2 in ANGPTL6 (3 patients), 1 in ADAMTS15 (1 patient) and 1 in TMEM132B (1 patient). In the affected family, prioritization of shared sequence variants yielded five novel putative risk genes. Based on predicted pathogenicity of identified variants, population genetics data and a high functional relevance for vascular biology, EDIL3 was selected as top candidate and screened in additional 37 individuals with UIA and/or aSAH: a further very rare EDIL3 sequence variant in two unrelated sporadic patients was identified.

Conclusions: Our data support a role of sequence variants in PCNT, RNF213 and THSD1 as susceptibility factors for cerebrovascular disease. The documented function in vascular wall integrity, the crucial localization of affected amino acids and gene/variant association tests suggest EDIL3 as a further valid candidate disease gene for UIA/aSAH.
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http://dx.doi.org/10.1007/s00415-020-09865-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419486PMC
September 2020

Cellular Importin-α3 Expression Dynamics in the Lung Regulate Antiviral Response Pathways against Influenza A Virus Infection.

Cell Rep 2020 04;31(3):107549

Viral Zoonosis - One Health, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany; Institute of Virology, University of Veterinary Medicine, Hannover, Germany. Electronic address:

Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections.
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http://dx.doi.org/10.1016/j.celrep.2020.107549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7172908PMC
April 2020

A prenatally disrupted airway epithelium orchestrates the fetal origin of asthma in mice.

J Allergy Clin Immunol 2020 06 15;145(6):1641-1654. Epub 2020 Apr 15.

Department of Obstetrics and Prenatal Medicine, Laboratory for Experimental Feto-Maternal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

Background: Prenatal challenges such as maternal stress perception increase the risk and severity of asthma during childhood. However, insights into the trajectories and targets underlying the pathogenesis of prenatally triggered asthma are largely unknown. The developing lung and immune system may constitute such targets.

Objective: Here we have aimed to identify the differential sex-specific effects of prenatal challenges on lung function, immune response, and asthma severity in mice.

Methods: We generated bone marrow chimeric (BMC) mice harboring either prenatally stress-exposed lungs or a prenatally stress-exposed immune (hematopoietic) system and induced allergic asthma via ovalbumin. Next-generation sequencing (RNA sequencing) of lungs and assessment of airway epithelial barrier function in ovalbumin-sensitized control and prenatally stressed offspring was also performed.

Results: Profoundly enhanced airway hyperresponsiveness, inflammation, and fibrosis were exclusively present in female BMC mice with prenatally stress-exposed lungs. These effects were significantly perpetuated if both the lungs and the immune system had been exposed to prenatal stress. A prenatally stress-exposed immune system alone did not suffice to increase the severity of these asthma features. RNA sequencing analysis of lungs from prenatally stressed, non-BMC, ovalbumin-sensitized females unveiled a deregulated expression of genes involved in asthma pathogenesis, tissue remodeling, and tight junction formation. It was also possible to independently confirm a tight junction disruption. In line with this, we identified an altered perinatal and/or postnatal expression of genes involved in lung development along with an impaired alveolarization in female prenatally stressed mice.

Conclusion: Here we have shown that the fetal origin of asthma is orchestrated by a disrupted airway epithelium and further perpetuated by a predisposed immune system.
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http://dx.doi.org/10.1016/j.jaci.2020.01.050DOI Listing
June 2020

Molecular profiling of an osseous metastasis in glioblastoma during checkpoint inhibition: potential mechanisms of immune escape.

Acta Neuropathol Commun 2020 03 9;8(1):28. Epub 2020 Mar 9.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany.

Peripheral metastases of glioblastoma (GBM) are very rare despite the ability of GBM cells to pass through the blood-brain barrier and be disseminated through the peripheral blood. Here, we describe a detailed genetic and immunological characterization of a GBM metastasis in the skeleton, which occurred during anti-PD-1 immune checkpoint therapy. We performed whole genome sequencing (WGS) and 850 K methylation profiling of the primary and recurrent intracranial GBM as well as one of the bone metastases. Copy number alterations (CNA) and mutational profiles were compared to known genomic alterations in the TCGA data base. In addition, immunophenotyping of the peripheral blood was performed. The patient who was primarily diagnosed with IDH-wildtype GBM. After the resection of the first recurrence, progressive intracranial re-growth was again detected, and chemotherapy was replaced by PD-1 checkpoint inhibition, which led to a complete intracranial remission. Two months later MR-imaging revealed multiple osseous lesions. Biopsy confirmed the GBM origin of the skeleton metastases. Immunophenotyping reflected the effective activation of a peripheral T-cell response, with, however, increase of regulatory T cells during disease progression. WGS sequencing demonstrated distinct genomic alterations of the GBM metastasis, with gains along chromosomes 3 and 9 and losses along chromosome 4, 10, and 11. Mutational analysis showed mutations in potentially immunologically relevant regions. Additionally, we correlated tumour-infiltrating lymphocyte and microglia presence to the occurrence of circulating tumour cells (CTCs) in a larger cohort and found a decreased infiltration of cytotoxic T cells in patients positive for CTCs. This study exemplifies that the tumour microenvironment may dictate the response to immune checkpoint therapy. In addition, our study highlights the fact that despite an effective control of intracranial GBM, certain tumour clones have the ability to evade the tumour-specific T-cell response and cause progression even outside of the CNS.
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http://dx.doi.org/10.1186/s40478-020-00906-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063778PMC
March 2020

Clonal Evolution after Allogeneic Hematopoietic Stem Cell Transplantation: The Case of Myelofibrosis.

Biol Blood Marrow Transplant 2020 07 5;26(7):e167-e170. Epub 2020 Mar 5.

Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

The significance of clonal evolution in myelofibrosis (MF) relapse remains poorly understood. Here we performed panel sequencing in paired samples of 30 patients with MF who relapsed after undergoing allogeneic hematopoietic stem cell transplantation (alloSCT). We identified a median of 2 mutations (range, 0 to 12) in a median of 2 genes (range, 0 to 8) before allo-SCT, along with a median of 2 mutations (range, 0 to 12) in 2 genes (range, 0 to 6) at relapse. Additional whole-genome sequencing (n = 6) did not elucidate additional molecular changes. Taken together, our data provide further evidence, here on MF, that clonal evolution after alloSCT is limited and that instead, alloSCT selects specific (sub)clones.
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http://dx.doi.org/10.1016/j.bbmt.2020.02.021DOI Listing
July 2020

The landscape of viral associations in human cancers.

Nat Genet 2020 03 5;52(3):320-330. Epub 2020 Feb 5.

Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, for which whole-genome and-for a subset-whole-transcriptome sequencing data from 2,658 cancers across 38 tumor types was aggregated, we systematically investigated potential viral pathogens using a consensus approach that integrated three independent pipelines. Viruses were detected in 382 genome and 68 transcriptome datasets. We found a high prevalence of known tumor-associated viruses such as Epstein-Barr virus (EBV), hepatitis B virus (HBV) and human papilloma virus (HPV; for example, HPV16 or HPV18). The study revealed significant exclusivity of HPV and driver mutations in head-and-neck cancer and the association of HPV with APOBEC mutational signatures, which suggests that impaired antiviral defense is a driving force in cervical, bladder and head-and-neck carcinoma. For HBV, HPV16, HPV18 and adeno-associated virus-2 (AAV2), viral integration was associated with local variations in genomic copy numbers. Integrations at the TERT promoter were associated with high telomerase expression evidently activating this tumor-driving process. High levels of endogenous retrovirus (ERV1) expression were linked to a worse survival outcome in patients with kidney cancer.
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http://dx.doi.org/10.1038/s41588-019-0558-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076016PMC
March 2020

Characterization of novel, recurrent genomic rearrangements as sensitive MRD targets in childhood B-cell precursor ALL.

Blood Cancer J 2019 11 29;9(12):96. Epub 2019 Nov 29.

Department of Pediatric Hematology and Oncology, University Medical Center Hamburg, 20246, Hamburg, Germany.

B-cell precursor (BCP) ALL carry a variety of classical V(D)J rearrangements as well as genomic fusions and translocations. Here, we assessed the value of genomic capture high-throughput sequencing (gc-HTS) in BCP ALL (n = 183) for the identification and implementation of targets for minimal residual disease (MRD) testing. For TRδ, a total of 300 clonal rearrangements were detected in 158 of 183 samples (86%). Beside clonal Vδ2-Dδ3, Dδ2-Dδ3, and Vδ2-Jα we identified a novel group of recurrent Dδ-Jα rearrangements, comprising Dδ2 or Dδ3 segments fused predominantly to Jα29. For IGH-JH, 329 clonal rearrangements were identified in 172 of 183 samples (94%) including novel types of V(D)J joining. Oligoclonality was found in ~1/3 (n = 57/183) of ALL samples. Genomic breakpoints were identified in 71 BCP-ALL. A distinct MRD high-risk subgroup of IGH-V(D)J-germline ALL revealed frequent deletions of IKZF1 (n = 7/11) and the presence of genomic fusions (n = 10/11). Quantitative measurement using genomic fusion breakpoints achieved equivalent results compared to conventional V(D)J-based MRD testing and could be advantageous upon persistence of a leukemic clone. Taken together, selective gc-HTS expands the spectrum of suitable MRD targets and allows for the identification of genomic fusions relevant to risk and treatment stratification in childhood ALL.
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http://dx.doi.org/10.1038/s41408-019-0257-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884523PMC
November 2019

DAMIAN: an open source bioinformatics tool for fast, systematic and cohort based analysis of microorganisms in diagnostic samples.

Sci Rep 2019 11 14;9(1):16841. Epub 2019 Nov 14.

Heinrich-Pette-Institute (HPI), Leibniz Institute for Experimental Virology, Research Group Virus Genomics, Hamburg, Germany.

We describe DAMIAN, an open source bioinformatics tool designed for the identification of pathogenic microorganisms in diagnostic samples. By using authentic clinical samples and comparing our results to those from established analysis pipelines as well as conventional diagnostics, we demonstrate that DAMIAN rapidly identifies pathogens in different diagnostic entities, and accurately classifies viral agents down to the strain level. We furthermore show that DAMIAN is able to assemble full-length viral genomes even in samples co-infected with multiple virus strains, an ability which is of considerable advantage for the investigation of outbreak scenarios. While DAMIAN, similar to other pipelines, analyzes single samples to perform classification of sequences according to their likely taxonomic origin, it also includes a tool for cohort-based analysis. This tool uses cross-sample comparisons to identify sequence signatures that are frequently present in a sample group of interest (e.g., a disease-associated cohort), but occur less frequently in control cohorts. As this approach does not require homology searches in databases, it principally allows the identification of not only known, but also completely novel pathogens. Using samples from a meningitis outbreak, we demonstrate the feasibility of this approach in identifying enterovirus as the causative agent.
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http://dx.doi.org/10.1038/s41598-019-52881-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856179PMC
November 2019

Extremophilic nitrite-oxidizing Chloroflexi from Yellowstone hot springs.

ISME J 2020 02 17;14(2):364-379. Epub 2019 Oct 17.

Department of Microbiology and Biotechnology, Universität Hamburg, Hamburg, Germany.

Nitrifying microorganisms occur across a wide temperature range from 4 to 84 °C and previous studies in geothermal systems revealed their activity under extreme conditions. Archaea were detected to be responsible for the first step of nitrification, but it is still a challenging issue to clarify the identity of heat-tolerant nitrite oxidizers. In a long-term cultivation approach, we inoculated mineral media containing ammonium and nitrite as substrates with biofilms and sediments of two hot springs in Yellowstone National Park (USA). The nitrifying consortia obtained at 70 °C consisted mostly of novel Chloroflexi as revealed by metagenomic sequencing. Among these, two deep-branching novel Chloroflexi were identified as putative nitrite-oxidizing bacteria (NOB) by the presence of nitrite oxidoreductase encoding genes in their genomes. Stoichiometric oxidation of nitrite to nitrate occurred under lithoautotrophic conditions, but was stimulated by organic matter. Both NOB candidates survived long periods of starvation and the more abundant one formed miniaturized cells and was heat resistant. This detection of novel thermophilic NOB exemplifies our still incomplete knowledge of nitrification, and indicates that nitrite oxidation might be an ancient and wide-spread form of energy conservation.
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http://dx.doi.org/10.1038/s41396-019-0530-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976673PMC
February 2020

De novo mutations in mitochondrial DNA of iPSCs produce immunogenic neoepitopes in mice and humans.

Nat Biotechnol 2019 10 19;37(10):1137-1144. Epub 2019 Aug 19.

Department of Surgery, Division of Cardiothoracic Surgery, Transplant and Stem Cell Immunobiology Lab, University of California, San Francisco, San Francisco, CA, USA.

The utility of autologous induced pluripotent stem cell (iPSC) therapies for tissue regeneration depends on reliable production of immunologically silent functional iPSC derivatives. However, rejection of autologous iPSC-derived cells has been reported, although the mechanism underlying rejection is largely unknown. We hypothesized that de novo mutations in mitochondrial DNA (mtDNA), which has far less reliable repair mechanisms than chromosomal DNA, might produce neoantigens capable of eliciting immune recognition and rejection. Here we present evidence in mice and humans that nonsynonymous mtDNA mutations can arise and become enriched during reprogramming to the iPSC stage, long-term culture and differentiation into target cells. These mtDNA mutations encode neoantigens that provoke an immune response that is highly specific and dependent on the host major histocompatibility complex genotype. Our results reveal that autologous iPSCs and their derivatives are not inherently immunologically inert for autologous transplantation and suggest that iPSC-derived products should be screened for mtDNA mutations.
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http://dx.doi.org/10.1038/s41587-019-0227-7DOI Listing
October 2019
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