Detection of SARS-CoV-2-RNA in post-mortem samples of human eyes

Abstract Purpose To detect SARS-CoV-2 RNA in post-mortem human eyes. Ocular symptoms are common in patients with COVID-19. In some cases, they can occur before the onset of respiratory and other symptoms. Accordingly, SARS-CoV-2 RNA has been detected in conjunctival samples and tear film of patients suffering from COVID-19. However, the detection and clinical relevance of intravitreal SARS-CoV-2 RNA still remain unclear due to so far contradictory reports in the literature. Methods In our study 20 patients with confirmed diagnosis of COVID-19 were evaluated post-mortem to assess the conjunctival and intraocular presence of SARS-CoV-2 RNA using sterile pulmonary and conjunctival swabs as well as intravitreal biopsies (IVB) via needle puncture. SARS-CoV-2 PCR and whole genome sequencing from the samples of the deceased patients were performed. Medical history and comorbidities of all subjects were recorded and analyzed for correlations with viral data. Results SARS-CoV-2 RNA was detected in 10 conjunctival (50%) and 6 vitreal (30%) samples. SARS-CoV-2 whole genome sequencing showed the distribution of cases largely reflecting the frequency of circulating lineages in the Munich area at the time of examination with no preponderance of specific variants. Especially there was no association between the presence of SARS-CoV-2 RNA in IVBs and infection with the variant of concern (VOC) alpha. Viral load in bronchial samples correlated positively with load in conjunctiva but not the vitreous. Conclusion SARS-CoV-2 RNA can be detected post mortem in conjunctival tissues and IVBs. This is relevant to the planning of ophthalmologic surgical procedures in COVID-19 patients, such as pars plana vitrectomy or corneal transplantation. Furthermore, not only during surgery but also in an outpatient setting it is important to emphasize the need for personal protection in order to avoid infection and spreading of SARS-CoV-2. Prospective studies are needed, especially to determine the clinical relevance of conjunctival and intravitreal SARS-CoV-2 detection concerning intraocular affection in active COVID-19 state and in post-COVID syndrome.

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Complete Genome Sequences for Two Strains of a Novel Fastidious, Partially Acid-Fast, Gram-Positive Corynebacterineae Bacterium, Derived from Human Clinical Samples

Genome Announcements ◽

10.1128/genomea.01462-15 ◽

2015 ◽

Vol 3 (6) ◽

Cited By ~ 4

Author(s):

Ainsley C. Nicholson ◽

Melissa Bell ◽

Ben W. Humrighouse ◽

John R. McQuiston

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Relevance ◽

Complete Genome ◽

Growth Characteristics ◽

Clinical Samples ◽

Whole Genome ◽

The Novel ◽

Genome Sequences ◽

Gram Positive

Here we report the complete genome sequences of two strains of the novel fastidious, partially acid-fast, Gram-positive bacillus “ Lawsonella clevelandensis ” (proposed). Their clinical relevance and unusual growth characteristics make them intriguing candidates for whole-genome sequencing.

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472P Whole genome sequencing of metastatic CRC reveals footprints from the past and present with future clinical relevance

Annals of Oncology ◽

10.1016/j.annonc.2020.08.583 ◽

2020 ◽

Vol 31 ◽

pp. S442

Author(s):

P.A.J. Mendelaar ◽

M. Smid ◽

J. Van Riet ◽

L. Angus ◽

H.J.G. Van de Werken ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Relevance ◽

Whole Genome ◽

The Past

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Whole Genome Sequencing Identifies Microdeletions Affecting TET2 and RUNX1 with Clinical Impact in Myeloid Malignancies

Blood ◽

10.1182/blood-2021-150646 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3672-3672

Author(s):

Constance Baer ◽

Wencke Walter ◽

Stephan Hutter ◽

Niroshan Nadarajah ◽

Wolfgang Kern ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Relevance ◽

Normal Karyotype ◽

Genetic Mechanism ◽

Patient Specific ◽

Whole Genome ◽

Myeloid Malignancies ◽

Ras Genes ◽

Genetic Mechanisms

Abstract Background: The current routine genetic work-up in hematological malignancies includes chromosome banding analysis (CBA) to detect complete or partial chromosomal deletions and fusions, and the identification of point mutations and small deletions or insertions by sequencing panels (max. length ~50 bp). Deletions of individual genes (e.g. IKZF1 in ALL) are only detected by specifically designed molecular tools. Therefore, those microdeletions might be overlooked by the current gold standard despite their clinical relevance. We established a bioinformatic pipeline to screen for microdeletions in whole genome sequencing (WGS) data of myeloid malignancies. Aim: (1) Screen for recurrent microdeletions in myeloid malignancies with a normal karyotype, and (2) characterize a patient specific profile of microdeletions in genes with known clinical and/or prognostic relevance. Patients and Methods: We analyzed 1356 cases (M/F: 778/578) of myeloid malignancies with a normal karyotype according to CBA (aCML: n=47; AML: n=251; CMML: n=165, mastocytosis: n=90; MDS: n=415, MDS/MPN-RS-T: n=69; MDS/MPN-U: n=42; MPN: n=250; PNH: n=27) using WGS. Median age was 71 [20-94] years. Amplification-free WGS was performed on the NovaSeq or HiSeq system with a median coverage of 103x (Illumina, San Diego, CA). Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and somatic copy number variant (CNV) discovery was performed with GATK (v 4.0.2.1), following best practice guidelines. Only gene overlapping CNV calls were considered for analysis (gene coordinates biomaRt (v 2.42.1), GRCh37 Ensembl). Results: On average, 38 genes per patient were partially or completely deleted and the size of the deletions ranged from 0.9 kb to 32 Mb (median 399 kb). The microdeletions affected a broad list of genes, but no gene was present in >5% of myeloid malignancies. As technical validation, we used 36 B-ALL samples (normal karyotype) and identified the known deletions of IKZF1 (42%); PAX5 (25%) and CDKN2A/CDKN2B (22%) with expected incidences. We focused on a patient-by-patient analysis of genes (n=47) with known clinical relevance in myeloid malignancies. We identified deleted genes in 46 out of 1356 patients (3.4%). In aCML 13% of patients had one of the above-mentioned genes deleted (6/47), in mastocytosis only 1% (1/90). The most frequently deleted genes were TET2 (20/1356, 1.5%) and RUNX1 (9/1356, 0.7%). Other deletions also affected transcription factors (e.g. GATA2) or epigenetic regulators (e.g. DNMT3A, figure 1). No deletion of splicing factors, RAS genes or cohesion complex regulators was observed. We found only two deletions of kinases, which are predominantly affected by activating mutations (both FLT3). Instead, the deletions in 41 patients involved genes with a known loss-of-function mutation profile in myeloid malignancies. This corresponds to 89% (41/46) of patients with microdeletions or 3% (41/1356) of all analyzed patients with myeloid malignancies. Microdeletions are thus another genetic element that can lead to loss of gene activity. Deletions and mutations are either alternative genetic mechanisms or co-operate as double hits to affect the same gene. We found additional mutations present in 18 of the 46 patients with microdeletions (39%, figure 1). The majority of these (n=14) involved TET2. TET2 mutations had a median variant allele frequency of 82% [9-100%] indicative of a mutation on the non-deleted allele. For the remaining genes (incl. RUNX1), deletions are predominantly an alternative genetic mechanism to mutations. For validation of WGS results we applied interphase FISH and identified 6/9 RUNX1 deletions. The remaining three microdeletions were only detectable by WGS and too small to be identified by FISH. Conclusions: (1) WGS data unrevealed a plethora of microdeletions, which can be an alternative genetic mechanism to mutations, but are not detected with today's standard diagnostic tools. (2) In the light of increasingly personalized therapy and diagnostics, all genetic mechanisms should be considered, which impact the function of clinically relevant genes. (3) Bioinformatic pipelines for WGS as a potential diagnostic tool in the near future should address microdeletions in genes with relevance for patients' diagnosis, prognosis and hopefully targeted treatment. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

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Resolution of a Clinical Dilemma with Whole Genome Sequencing, and Discovery of a New Mechanism for Generating PML-Rara: Insertional Fusion

Blood ◽

10.1182/blood.v116.21.2755.2755 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2755-2755

Author(s):

John S. Welch ◽

Li Ding ◽

Ken Chen ◽

David E. Larson ◽

Shashikant Kulkarni ◽

...

Keyword(s):

Bone Marrow ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Relevance ◽

Chromosomal Rearrangement ◽

Chromosome 15 ◽

Low Grade ◽

Whole Genome ◽

Rt Pcr ◽

Oncogenic Form

Abstract Abstract 2755 We describe a difficult diagnostic case of t(15;17)-negative acute promyelocytic leukemia (APL). A 39 year-old woman presented with pancytopenia and low grade DIC. Bone marrow biopsy revealed AML with promyelocytic features. She was treated with Cytarabine, Daunorubicin and ATRA. However, ATRA was discontinued after FISH revealed a possible RARA-PML, but no PML-RARA fusion (one fusion, one RARA and two PML signals), and cytogenetics did not demonstrate a translocation involving chromosome 15 or 17. In fact, her cytogenetics revealed a complex pattern that predicted poor prognosis (46 XX del(9)(q12q32),del(12)(q12q21)[6]/46,idem,-6,-16,add(16)(p13.2),+2 mar[13]/46 XX[1]). Following reinduction, she entered complete remission and was empirically consolidated with arsenic. RT-PCR for PML-RARA was not performed at diagnosis, and was negative at the time of consultation in remission. Her complex cytogenetics and uncertain FISH status posed a diagnostic and therapeutic dilemma that could only be resolved by whole genome sequencing in the time frame required for a clinical decision to be made (i.e. allogeneic transplantation vs. ATRA-based consolidation). DNA was therefore generated from bone marrow (cryopreserved at the time of diagnosis) and a skin sample (obtained in remission), and subjected to massively parallel sequencing using paired-end reads (Mardis et al, NEJM 2009). We generated 187 billion bp (tumor) and 200 billion bp (skin) of sequence, corresponding to 43.7x and 46.8x haploid coverage (99.76% and 99.74% diploid coverage) for the respective samples. Within six weeks of sample receipt, validated results were available. We confirmed the del(9)(q12q32) and del(12)(q12q21) somatic events and identified a novel oncogenic form of chromosomal rearrangement, an insertional fusion: 77 Kb of chromosome 15 (chr15:72027045–72104108 containing LOXL1 exon 6 through PML exon 3) were inserted en bloc into RARA intron 2 (chr17:35742678–35742683). This event resulted in the expression of PML-RARA (bcr3 isoform) and two novel fusion transcripts (RARA-LOXL1 and LOXL1-PML), which were all successfully amplified by RT-PCR and sequenced. The RARA-LOXL1 and LOXL1-PML fusions both created stop codons shortly after the fusion events. Re-evaluation of the FISH results revealed that the insertion generated a fusion signal, while loss of 77 Kb from the PML locus did not prevent binding of the 239 Kb commercial PML probe to chromosome 15 (thus generating 1 fusion, 1 RARA and 2 PML signals). These signals represented the PML-RARA insertional fusion event, not the RARA-PML translocation that was originally reported. We further identified and validated deletions on chromosomes 12 (60 Mb), 14 (22 Kb) and 19 (30 Kb) and non-synonymous, somatic single nucleotide variants (SNVs) in the coding regions of ZNF687, DYTN, C3orf54, CH3D19, SLC35A4, GPRC6A, ZFHX4, PTK2, PITPNM1, DEGS2, PCSK2, CDC45L, although the clinical relevance of these deletions and point mutations is not yet known. After validating PML-RARA bcr3 expression, a decision was made to consolidate the patient with an ATRA-containing regimen. Using whole genome sequencing with paired end reads, we have identified a novel oncogenic form of chromosomal rearrangement, an insertional fusion. Similar insertional events may occur in other loci. Small structural events (under a few megabases in size) are often undetectable by conventional cytogenetics and FISH, and are expected to be invisible to standard break-apart probes (commonly used to evaluate the RARA and MYC loci). This case highlights the clinical relevance of whole genome sequencing for informing diagnostic and therapeutic decisions that must be made within weeks after sample acquisition. Disclosures: Off Label Use: Decitabine, Arsenic and Ascorbic acid for the treatment of AML. DiPersio:Genzyme: Honoraria. Westervelt:Novartis: Honoraria; Celgene: Honoraria, Speakers Bureau.

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