Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

The Telomerase Reverse Transcriptase Subunit from the Dimorphic Fungus <i>Ustilago maydis</i>

<div><p>In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus <i>Ustilago maydis</i>. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting <i>trt1</i> had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in <i>est2</i> budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of <i>trt1</i> crosses <i>in planta</i> suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.</p></div

Telomere repeat amplification protocol (TRAP) analysis in <i>U. maydis</i>.

<p>Telomerase activity in wild-type and mutant strains was determined. The absorbance data were used to construct a graphical representation of the telomerase activity for the sporidia of <i>U. maydis</i> strains (either wild-type or <i>trt</i>^-). Tumor cells derived from the 521×520 cross and a plant control were included to evaluate and detect telomerase activity. The medians of the telomerase-positive control cells (HEK293) and the 521 wild-type strain were significantly different from the median of the treated negative controls (P<0.05); however, no significant differences were detected between the negative controls and the <i>trt1</i>-disrupted mutants. The samples heated to 85°C are indicated with Δ, and the RNase-treated samples are designated as RNase. Telomerase activity was also determined in tumors and maize leaves.</p

Determination of telomerase activity in <i>U. maydis</i> strains.

<p>The average absorbance values yielded by the telomerase activity in cell extracts are numerically expressed and shown in the chart. The absorbance for each sample was calculated according to the manufacturer's instructions and is shown. Heat- (85°C) and RNase-treated samples were used as negative controls. Telomerase activity was measured under the same conditions in the positive controls and tested samples. Telomerase activity was measured only in the mutant trt1-1; telomerase-negative samples (either from mutants, tumors, or plants) were not treated with heat or RNase, and their activity was not determined (N.D.). All of the experiments were performed at least three times.</p><p>* Media of at least three repetitions.</p><p>Determination of telomerase activity in <i>U. maydis</i> strains.</p

Structure of the <i>U. maydis</i> um11198 locus.

<p>The illustrative representation of the locus encoding the putative telomerase reverse transcriptase subunit (Trt1) of <i>U. maydis</i> is shown. (A) The open reading frame is depicted as a box, and the TERT domains are colored. The thin black lines represent the non-coding sequences located up- and downstream of the gene. (B) The conserved GQ (blue), RBD (red), and TR (green) domains of Trt1 are indicated above each highlighted alignment. The conserved residues are colored as in A. The sequences are from the representative organisms <i>Homo sapiens</i> (accession NP_937983.2), <i>Arabidopsis thaliana</i> (accession AF172097_1), and <i>Saccharomyces cerevisiae</i> (accession AAB64520.1), where the motif E does not align with other TERTs (asterisk). The aligned sequences used to define the motifs include at least 12 species, but only the representative organisms are shown.</p

<i>U. maydis</i> strains used.

<p>^*Partially characterized.</p><p><i>U. maydis</i> strains used.</p

Analysis of the effects of <i>trt</i>+ restoration in pTrt1 <i>U. maydis</i> transformants.

<p>The telomere length distribution in the telomerase-deficient strain W204 was assessed by Southern blotting TRF after reintroduction (200 doubling periods) of <i>tert1</i> in the pTrt1 <i>U. maydis</i> transformants as described above. (A) The TRF hybridization pattern of parental 521 (lane 1), 520 (lane 2), <i>trt1</i>-disrupted mutants trt1-1 (lane 3) and trt1-2 (lane 4) strains, the progeny derivative W204 (lane 5), and five of its W204-derived clones (T1 to T5, lanes 6 to 10) were analyzed using telomere sequences (TTAGGG) that were ³²P- labeled at 17 kBq/ml as probes. (C) The filter was stripped and re-hybridized to TR-p + TR-d sequences ³²P- labeled as a probe. The strain names are shown above the autoradiography. A molecular weight marker is shown on the left.</p

One-step disruption and recovery of <i>trt^-</i> mutants.

<p>(A) The putative <i>trt1</i> gene is depicted as a black box (upper portion). The assembly containing the <i>hph</i> gene, which was used as selectable marker, is depicted as a light-gray box. The names of the primers used for the PCR assays and the lengths of the amplified products are indicated in the boxes below. The lines are to scale. (B) Multiplex-PCR amplification products of the <i>U. maydis</i> wild type strain 521, the 520 strain, trt1-1 and trt1-2 transformants after <i>trt1</i> disruption, and two clones of the 08 ectopic recombinant (upper gel). Disruption was verified via amplification of the fragment spanning part of the hygromycin-resistance gene and the <i>trt1</i> promoter (lower gel). EE indicates the DNA amplified using EST2 FDD1 and EST2 RDD1 (904 bp, wild-type pattern), and EH indicates the DNA amplified using EST2 FDD1 and HyR RDD1 (1,286-bp, <i>trt1</i>-disrupted or non-homologous recombinant pattern). E2H indicates DNA amplified with EST2FDD2 and HyR RDD1 (1,941-bp disrupted pattern). A molecular weight marker is shown on the left, and the strain name is shown in the top. (C) The phenotype of one disrupted strain, named <i>trt1-1</i>, was evaluated after 12, 60, 156, and 180 doubling periods. Small, dry, irregular, slightly hyper-pigmented colonies with low replicative potential were observed after 180 doubling periods. The number of doubling periods is shown on each quarter of the petri dish.</p

Terminal restriction fragment (TRF) analysis of a <i>trt1</i>-disrupted <i>U. maydis</i> mutant.

<p>(A) Samples of total DNA from the <i>U. maydis</i> 521 strain (150 ng) and trt1-1 mutant clone (500 ng) were digested with <i>Pst</i>I and quantified. The wild-type 521 strain was used as a control. A 36-generation series of samples was acquired from the trt1-1 mutant culture at the following population doubling times: 76 (lane 1), 112 (lane 2), 148 (lane 3), 184 (lane 4), 220 (lane 5), 256 (lane 6), 292 (lane 7), and 328 (lane 8). The DNA samples were hybridized to a TTAGGG probe as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109981#s4" target="_blank">Materials and Methods</a>. (B) The nylon membrane was stripped and re-hybridized to UT4-a probe, an <i>Eco</i>RI/<i>Bam</i>HI fragment from the subtelomeric <i>UTASa</i> sequence, at high stringency conditions. The sizes (kb) are indicated on the left, and the arrows indicate the DNA bands showing changes in mobility or appearance.</p

Extrachromosomal <i>trt1</i> complementation and growth rate restoration.

<p>After restoring the replicative potential by <i>trt1</i> reintroduction, the growth rate was analyzed in the transformants with the same method used for the non-transformed controls. The strains analyzed are shown on the left, and the plasmids used to transform these strains are indicated at the top. In parentheses, minutes were changed to hours.</p><p>Extrachromosomal <i>trt1</i> complementation and growth rate restoration.</p