Shorter telomere lengths in patients with severe COVID-19 disease

Pathologies in COVID-19 patient cohort

In order to assess the potential impact of telomere length on pathologies associated to COVID-19 disease, we obtained both DNA and mononuclear cells from peripheral blood samples from patients hospitalized at the IFEMA field hospital in Madrid, which was constructed to treat COVID-19 patients. A total of 61 female and 28 male patients of ages ranging from 29 to 85 years old were included in the study (Table 1). The patient cohort had different severity of pathologies and received the treatments indicated in Table 1. None of the patients included in this study died as a consequence of the COVID-19 disease.

In order to correlate patient severity with telomere length, we first grouped the patients according the a severity score ranging from 1 to 4, with severity score 1 in the case of patients with low fever and cough but without any radiological features of pneumonia to patients with severity score of 4 in the cases of patients with features of Acute Respiratory Distress Syndrome (ARDS) requiring mechanical ventilation along with presence of multiorgan dysfunction failure, metabolic acidosis and coagulation dysfunction (Materials and Methods).

Determination of telomere length in COVID-19 patients

In order to determine telomere length in our patient cohort, peripheral blood was extracted from the arm from the different patients and used to measure telomere length by three independent techniques (Materials and Methods). First, we determined telomere length in DNA extracted from peripheral blood by using both the Southern blotting-based Telomere Restriction Analysis (TRF; see [38]) and the quantitative-PCR (qPCR) telomere length analyses [45]. In addition to these two technologies based on DNA samples, we also measured telomere length on fresh peripheral blood mononuclear cells by using the more precise high-throughput quantitative fluorescence in situ hybridization (HT Q-FISH) previously described by us [34, 35, 46], which allows determination of individual telomere fluorescence signals using tens of thousands of cells from a single patient. The fact that HT Q-FISH can determine individual telomere fluorescence spots in interphasic nuclei, each spot usually formed by several clustered telomeres, allows to the determine the abundance of very short telomeres.

We observed a very significant correlation between the telomere length measurements obtained by the three different techniques (Figure 1A), thus indicating the robustness of the data on telomere length obtained in our patient cohort. Given the good correlation of the telomere length data obtained with the three technologies, we decided to perform the rest of the analysis with the telomere length data obtained by HT Q-FISH, as it measures telomeres in a single cell manner and it also allows to measure individual telomere spots within single nuclei.

The rate of telomere shortening in the patient cohort was of 77 bp/year (Figure 1B). This rate of telomere shortening is in the range previously published by us and others [45–48]. In agreement with telomere shortening with increasing patient age, we also observed an increase in the abundance of short telomeres (ie, telomere fluorescence spots corresponding to less than 3 kb of telomere length) which increased at a rate of 8.8 % per year (Figure 1B).

When we analyzed the data segregated by gender, linear regression of telomere length data in COVID-19 female patients showed that their telomeres were consistently longer than those of male patient at all age ranges, as well as they showed a lower percentage of short telomeres compared to male patients (Figure 2A–2D), also in agreement with previous findings [46]. Again, when segregated by gender, the rates of telomere shortening were in a range of 70-80 bp/year (Figure 2A). Similarly, the increase in the percentage of short telomeres with age was also similar in both genders (Figure 2A, 2B).

The fact that COVID-19 female patients had longer telomeres than men patients at different age ranges is in line with the fact that female COVID-19 patients show a lower mortality than males (see COVID-19 Sex-Disaggregated Data Tracker available at: http://globalhealth5050.org/covid19).

Age and telomere length correlate with COVID-19 severity

In order to assess whether short telomeres correlated with the severity of COVID-19 disease, we used a Pearson correlation analysis between the mean telomere length or the percentage of short telomeres (<3 kb) as determined by the HT Q-FISH technique, and either age or the severity score of the different COVID-19 patients ranging from 1 (less severe) to 4 (more severe) (see Materials and Methods).

As expected, we observed a significant inverse correlation between mean telomere length (TL) and age of the COVID-19 patients (r=-0.3985; p=0.0001; Figure 3A). We also observed a significant direct correlation between percentage of short telomeres (ie, telomeres < 3Kb) and patient age (r=0.285; p=0.0067; Figure 3B). Thus, these findings confirm a significantly higher incidence of short telomeres with increasing age in the COVID-19 patients. We also observed an inverse correlation between mean telomere length (TL) and the severity score of the COVID-19 patients when using HT Q-FISH (r=-0.1752; p=0.1026; Figure 3A) and a direct correlation between the percentage of short telomeres (ie, telomeres < 3Kb) and the severity score (r=0.1454; p=0.1766; Figure 3B), although these correlations did not reach statistical significance. To further analyze this, we performed similar analysis with the telomere length data obtained by TRF and by PCR (Figure 3C, 3D). Again, we confirmed a significant inverse correlation between mean telomere length (TL) and age of the COVID-19 patients by TRF (r=-0.4675; p<0.0001; Figure 3C) as well as by PCR (r=-0.405; p=0.0001; Figure 3D) techniques. Importantly, by these two DNA-based techniques, the correlation between telomere length and COVID-19 severity reached statistical significance (TRF: r=-0.3119, p=0.004; Figure 3C; PCR: r= -0.2308, p=0.036; Figure 3D.

Finally, we also observed a significant direct correlation between age of the COVID-19 patients and the severity score of the disease (r=0.2299; p=0.0312; Figure 4A, 4B). Furthermore, we observed an inverse correlation between age and mean telomere length (TL) and a direct correlation between age and the percentage of short telomeres when using HT Q-FISH (Figure 4A, 4B).

Together, these findings suggest significant correlations of age as well as telomere length with COVID-19 severity.

Higher severity of COVID-19 disease in patients in the lower percentiles of telomere length

The findings suggest that COVID-19 patients with shorter telomere length may have a higher risk of more severe pathologies. To further test this, we divided the patients in quartiles according to either their mean telomere length or their percentage of short telomeres using the telomere signal fluorescence data obtained by HT Q-FISH. We observed that patients in the lower quartile of mean telomere length (<25%) had a higher severity score (p=0.06; Figure 5A). Similarly, the patients in the higher quartile of percentage of short telomeres had significantly higher severity scores of the disease (p=0.049; Figure 5B).

Different rates of telomere shortening in patients with different severity scores

As patients in the lower quartile of telomere length have a significantly higher risk of severe COVID-19 pathologies, we set to investigate whether the rates of telomere shortening in higher severity score patients were significantly higher than in the lower severity score patients. To this end, we pooled together the patients in “mild-moderate” and “severe-acute” severity groups. We found that patients with a “severe-acute” diagnosis showed a significantly faster rate of telomere shortening compared to the “mild-moderate” diagnosis as determined by HT Q-FISH (p=0.024; Figure 6A). Of note, patients with “severe-acute” COVID-19 disease have shorter telomeres along all age groups compared to patients with “mild-moderate” COVID-19 disease. Similarly, we found an increased rate of accumulation of short telomeres in patients with a “severe-acute” diagnosis compared to patients with a “mild-moderate” severity score (p=0.08; Figure 6B).

In order to address whether this association between Covid-19 severity and short telomeres was independent of the age, we grouped the patients in different age groups (below 40 years of age; between 40-49; 50-59; 60-69 and over 70 years of age) and compared the fluorescence of individual telomere foci in patients showing either “mild-moderate” or “severe-acute” COVID-19 severity. We found that for age groups above 60 years of age, telomeres were shorter in the groups with “severe-acute” severity compared to “mild-moderate” severity (Figure 6C).

Patients

In this study participated a total of 89 patients (61 female and 28 male patients of ages ranging from 29 to 85 years old) from the IFEMA field hospital installed due to the emergency situation in Madrid, Spain. All these samples were donated to CNIO BioBank which allows their use for biomedical analyses under the existing law requirements in Spain.

Blood samples

A total of 8 ml of blood were collected from the arm of each patient in heparin tubes and 4 ml in EDTA tubes and shipped within less than 24h to the DNA National Bank at Salamanca University, where they were immediately processed at a biosafety level (BSL) 3 (BSL-3) biocontainment level. Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll gradient and frozen in 90% FBS (v/v) supplemented with 10% (v/v) DMSO into a number of aliquots ranging from 1 to 3, according to cell number. PBMCs were stored in vapor phase-nitrogen.

Genomic DNA was extracted directly from blood samples using the Real Blood DNA Kit and stored long-term in TE at -20° C.

Q-PCR Assay to measure average telomere length

Telomere length was measured in genomic DNA isolated from blood samples. We used a modified monochrome multiplex quantitative polymerase chain reaction (PCR) method already described [45]. Briefly, each reaction included SYBR Green I (Promega), telomere primer pair telg (5´-ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGTGT-3´) and telc (5´-TGTTAGGTATCCCTATCCCTATCCCTATCCCTATCCCTAACA-3´) (final concentrations 900nM each), albumin primer pair albu (5´-CGGCGGCGGGCGGCGCGGGCTGGGCGGaaatgctgcacagaatccttg-3´) and albd (5´-GCCCGGCCCGCCGCGCCCGTCCCGCCGgaaaagcatggtcgcctgtt-3´) (final concentrations 900nM each) and 20 ng of genomic DNA. The Applied Biosystems QuantStudio 6 Flex Real-Time PCR System was used. The thermal cycling profile was Stage 1: 15 min at 95° C; Stage 2: 2 cycles of 15 s at 94° C, 15 s at 49° C; and Stage 3: 32 cycles of 15 s at 94° C, 10 s at 62° C, 15 s at 74° C with signal acquisition, 10 s at 84° C, 15 s at 88° C with signal acquisition. To serve as a reference for standard curve calculation, HeLa cells were serially diluted and qPCR performed as described above. After thermal cycling was completed, the QuantStudio 6 software was used to generate standard curves and Ct values for telomere signals and reference gene signals. The average telomere length was termed T/S ratio. Finally, T/S ratios were converted into kb by external calibration with the K562 (6.5 kb), CCRF-CEM (7.5 kb), Jurkat (11.5 kb) and HeLa1211 (24 kb) cell lines.

Terminal restriction fragment analysis

Mean telomere length by Telomere Restriction Fragment (TRF) was determined using the method already described [38]. Briefly, genomic DNA was digested by MboI and separated by gel electrophoresis in 0.5X TBE maintained at 14° C, using a CHEF DR-II pulsed-field apparatus (BioRad) for 14 h at 5 V/cm at a constant pulse time of 0.5 s. The gel was transferred to a nylon membrane (Hybond-XL, GE Healthcare) and probed with a ³²P-labeled telomeric probe (TTAGGG)n (a gift from T. de Lange). Mean TRF lengths were determined using an ImageQuant TL.

HT Q-FISH

Clear bottom black-walled 96-well plates (Greiner, Longwood, FL) were precoated with a 0.001% (wt/vol) poly-L-lysine solution (Sigma-Aldrich, St. Louis, MO) for 1h at 37° C. Poly-L-lysine was removed and wells rinsed with RPMI before cell addition (75,000–150000 lymphocytes/well).

PBMCs were thawed in complete RPMI 1640 growth media supplemented with 10% FBS (v/v) and seeded at a concentration of 100000 cells/ well in triplicate wells per sample. Cells were left to adhere to the plate for 1 h at 37C in incubator with 95% humidity, 5% CO₂. Plates were then removed from incubator and then fixed at room temperature (RT) by slowly filling up the wells with 200 ul methanol/acetic acid (3/1, vol/vol) and incubated for 10 to 15 min. The solution was removed, and this was repeated 2 more times, leaving the last fixative volume up for a total of 1 h fixation. Plates were then moved to -20 until processed for high-throughput quantitative FISH (HT Q-FISH).

We performed HT Q-FISH as described before [46]. Briefly, the plates were removed from -20, the fixative solution removed, and the plates were dried on a hot plate at 37° C overnight, followed by rehydration with 200 μL of PBS. Cells were fixed with 200 μL of 4% formaldehyde in PBS for 2 min at room temperature (RT) and washed 3 times for 5 min with PBS. Prewarmed pepsin solution (100 mL of H₂O, 100 μL of 37% HCl [10.1 M HCl], and 100 mg of pepsin [Sigma-Aldrich; catalog no. P7000-25G]) was used to degrade cell walls for 15 min at 37° C followed by 2 washing steps of 5 min with 200 μL of PBS. The cells were then dehydrated with sequential 5-min 70%, 90%, and 100% ethanol steps. The plates were dried 1 h at 37° C. Next, 50 μL of the hybridization solution containing the Tel-Cy3 PNA probe was added to the plates (95 μL of 1 M Tris, pH 7.0, 812 μL of MgCl₂ solution [25 mM MgCl₂, 9 mM citric acid, 82 mM Na₂HPO₄], 6.65 mL of deionized formamide, 475 μL of blocking reagent [10 g of blocking reagent (Boehringer; catalog no. 1093 657) dissolved with heat in 100 mL of maleic acid buffer, pH 7.5 (100 mM maleic acid, 150 mM NaCl)], 1.28 mL of H₂O, and 190 μL of Tel-Cy3 PNA probe [5 μg lyophilized Cy3-(C3TA2)3 PNA probe (Panagene) diluted in 200 μL of H₂O]). Plates were then sealed with aluminum foil lids. The DNA was denatured by heating the plate on a hot plate at 85° C for 5 min and left to incubate for 2 h at RT in the dark. The plates were rinsed and washed in plate shaker with wash solution 1 (10 mM Tris-HCl pH 7, 70% Formamide, 0.10% BSA in H₂O) for 30 min, followed by 3 washes of 5 min each with wash solution 2 (TBS [Tris-buffered saline, pH 7.0] with 0.08% Tween 20). Nuclei staining was performed incubating for 10 min with TBST containing 1 μg/mL DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride; Life Technologies; catalog no. D-1306). Then the plates were washed 1 × 5 min with PBS and stored at 4° C in the dark. Images from the plate were then acquired by HT microscopy within 48 h.

High-throughput microscopy

Quantitative image acquisition was performed on an Opera High Content Screening System (PerkinElmer) 40×/0.9 N.A. water-immersion objective. UV and 561 nm excitation wavelengths were used to detect DAPI and Cy3 telomeric signals, respectively and 60 independent images were captured at different positions of each well. Images were analyzed with Acapella Image analysis software (PerkinElmer). Data were analyzed with SPSS (IBM) and Excel (Microsoft). Telomere fluorescence values were converted into kilobases by external calibration with the CCRF-CEM (7.5 kb), L5178Y-S (10.2 kb), L5178Y-R (79.7 kb) and Jurkat (11.5 kb) cell lines.

Criteria for the diagnosis of COVID-19

Depending on the clinical features of COVID-19, patients are generally divided as mild, moderate, severe and acute.

1. Mild COVID-19: low-grade fever, cough, malaise, rhinorrhea, sore throat with or without hemoptysis, nausea, vomiting, diarrhea, but without any radiological features of pneumonia and absence of mental changes.

2. Moderate COVID-19: fever, respiratory symptoms including dry cough and shortness of breath that may emerge along with the radiological features.

3. Severe COVID-19: dyspnea, respiratory frequency ≥30/minute, blood oxygen saturation ≤93%, PaO2/FiO2 ratio <300, and/or lung infiltrates >50% of the lung field within 24–48 h.

4. Acute COVID-19: usually develops after 7 days in patients with mild/moderate/severe COVID-19 with features of Acute respiratory distress syndrome (ARDS) requiring mechanical ventilation along with presence of multiorgan dysfunction failure, metabolic acidosis and coagulation dysfunction.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.