Since early 2020 SARS-CoV-2 infectious disease (COVID-19) has been responsible for more than 300.000 deaths across the globe.1 Advanced age and previous comorbidities are clearly related to the development of severe forms of the disease (sCOVID) and an increased mortality risk.2,3 However young healthy subjects with sCOVID are also admitted to intensive care units (ICU) and die, suggesting that individual variations and/or genetic predisposing factors might play a role in modifying the clinical course and severity of the disease.4
sCOVID-19 is characterized by fever, bilateral pneumonia, lymphopenia, hyperferritinemia, elevated acute phase reagents and cytokine storm, altogether conforming a hyperinflammation scenario similar to that in secondary hemophagocytic lymphochystiocytosis (sHLH), also known as macrophage activation syndrome.5 In adults, sHLH is mostly triggered by viral infections and approximately 50% of patients experience pulmonary disease.6 In contrast, familial HLH (fHLH) is genetically determined by mutations in genes coding for proteins related to lymphocyte cytotoxicity such as perforin (PRF1 gene). Studies in juvenile idiopathic arthritis or systemic lupus erithematosus patients show that up to 40% of individuals suffering sHLH carry heterozygous mutations in fHLH genes. In a fatal influenza A (H1N1) series, 36% of patients carried one or several mutations in fHLHrelated genes.7,8 These findings suggest an important, not yet totally recognized overlap between primary and secondary forms of HLH.
It has been previously observed that the highly prevalent, fHLH-associated c.272C>T variant (p.A91V; rs35947132) in the PRF1 gene impairs the processing to the active form of perforin protein.9 Published reports associate this variant with immune diseases but it has not been validated as pathological in larger cohorts.10 The A91V PRF1 gene translates into a protein with reduced stability and abnormal trafficking which associates with a significant decrease of NK-cell cytotoxicity.11,12 Previous studies reported higher prevalence of the A91V variant in HLH patients.13,14 It is reasonable to think that perforin bearing the A91V change could be related to suboptimal activation and effector capacities of CD8 and/or natural killer (NK) cells. In the context of a viral infection, the correct function of these cells is required to contain the viral replication, clear the virus and overcome the infection. Ineffective killing of SARS-CoV-2 infected cells might lead to a sustained activation of lymphocytes and macrophages contributing to the cytokine storm and hyperinflammation that characterizes sCOVID-19.
Based on the above premises, we hypothesized that the fHLH-associated A91V PRF1 variant is prevalent in patients suffering severe forms of COVID-19. We therefore tested for the A91V PRF1 variant in all sCOVID-19 patients in the ICU of our hospital on a random day (March 27). Exon 2 of the PRF1 gene coding region was amplified using PCR. PCR products were purified and sequenced as previously reported.15 Elderly and patients with comorbidities were excluded. Twenty-two previously healthy patients between the age of 24-52 years were identified: 17 of 22 males; 14 of 22 Latin- American, 7of 22 Spanish and 1 of 22 Polish.
Among the studied patients, 2 of 22 showed A91V PRF1 in heterozygosis (allele frequency of 0.045). According to the Genome Aggregation Database (gnomAD gnomad.broadinstitute.org), the calculated A91V PRF1 variant frequency in European plus Latino population is 0.031. Considering that no A91V-positive patients were detected among the Latin-American patients in intive care, the allele frequency found in our Europeans COVID-19 cohort was 0.125, almost 3-times higher than that described for Europeans in gnomAD (0.046).
After 6 weeks, 17of 20 A91V-negative patients had been discharged, 2 of 20 continued hospitalization with significant clinical improvement without ventilator requirement and 1 of 20 had died. Remarkably, both A91V-positive patients died. In these patients we calculated the value of the HScore, a previously validated score which includes the most important variables independently associated with sHLH and helps to form an accurate diagnosis of HLH. HScore values higher than 169 are considered positive for HLH, with a sensitivity of 93% and specificity of 86%.16 Both patients showed a high HScore for HLH (188 and 175),5 a shorter time from the disease onset to ICU admission (0 and 6 vs. 9.36 days on average) and more severe initial radiological findings (Table 1). Clinically, our A91V-positive patients had high fever associated with the respiratory symptoms. The HLH-related laboratory parameters triglycerides, fibrinogen, ferritin and aspartate aminotransferase were markedly elevated in both subjects, even while receiving immunosuppressive therapy. Unfortunately, because of the pandemic situation and rapid death of both patients, functional studies with cell samples could not be performed.
In conclusion, in our young sCOVID-19 patient cohort, A91V PRF1 was prevalent. A defective A91V PRF1 may translate into suboptimal lymphocyte cytotoxicity and ineffective SARS-CoV-2 clearance, favoring the progress to sCOVID-19 with an HLH-like clinical phenotype and high mortality. Our observation merits further investigations to assess the specific influence of this variant in COVID-19 clinical course. International collaborative efforts are needed to elucidate the role of genetics in COVID-19.
this work was supported by FIS-Instituto de Salud Carlos III, Grant Number: COV20/00181. This study was approved by the Institutional Ethical Board (20/167).
we thank the patients and their families, lab technicians and other health care providers of the Hospital 12 de Octubre for their participation in this research.
- Novel Coronavirus (2019-nCoV) situation reports-117 (May 2020). World Health Organization (WHO).Publisher Full TextGoogle Scholar
- Zhou F, Yu T, Du R. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study [published correction appears in Lancet. 2020; 395(10229):1038. https://doi.org/10.1016/S0140-6736(20)30566-3Google Scholar
- Yang J, Zheng Y, Gou X. Prevalence of comorbidities and its effects in patients infected with SARS-CoV-2: a systematic review and meta-analysis. Int J Infect Dis. 2020; 94:91-95. https://doi.org/10.1016/j.ijid.2020.03.017Google Scholar
- DeBiasi RL, Song X, Delaney M. Severe COVID-19 in children and young adults in the Washington, DC Metropolitan Region. J Pediatr. 2020; 223:199-203. https://doi.org/10.1016/j.jpeds.2020.05.007Google Scholar
- Mehta P, McAuley DF, Brown M. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet. 2020; 395(10229):1033-1034. https://doi.org/10.1016/S0140-6736(20)30628-0Google Scholar
- Seguin A, Galicier L, Boutboul D, Lemiale V, Azoulay E.. Pulmonary involvement in patients with hemophagocytic lymphohistiocytosis. Chest. 2016; 149(5):1294-1301. https://doi.org/10.1016/j.chest.2015.11.004Google Scholar
- Schulert GS, Zhang M, Fall N. Whole-exome sequencing reveals mutations in genes linked to hemophagocytic lymphohistiocytosis and macrophage activation syndrome in fatal cases of H1N1 influenza. J Infect Dis. 2016; 213(7):1180-1188. https://doi.org/10.1093/infdis/jiv550Google Scholar
- Crayne CB, Albeituni S, Nichols KE, Cron RQ. The immunology of macrophage activation syndrome. Front Immunol. 2019; 10:119. https://doi.org/10.3389/fimmu.2019.00119Google Scholar
- Trambas C, Gallo F, Pende D. A single amino acid change, A91V, leads to conformational changes that can impair processing to the active form of perforin. Blood. 2005; 106(3):932-937. https://doi.org/10.1182/blood-2004-09-3713Google Scholar
- Voskoboinik I, Lacaze P, Jang HS. Prevalence and disease predisposition of p.A91V perforin in an aged population of European ancestry. Blood. 2020; 135(8):582-584. https://doi.org/10.1182/blood.2019003487Google Scholar
- Voskoboinik I, Sutton VR, Ciccone A. Perforin activity and immune homeostasis: the common A91V polymorphism in perforin results in both presynaptic and postsynaptic defects in function. Blood. 2007; 110(4):1184-1190. https://doi.org/10.1182/blood-2007-02-072850Google Scholar
- House IG, Thia K, Brennan AJ. Heterozygosity for the common perforin mutation, p.A91V, impairs the cytotoxicity of primary natural killer cells from healthy individuals. Immunol Cell Biol. 2015; 93(6):575-580. https://doi.org/10.1038/icb.2015.1Google Scholar
- Busiello R, Fimiani G, Miano MG. A91V perforin variation in healthy subjects and FHLH patients. Int J Immunogenet. 2006; 33(2):123-125. https://doi.org/10.1111/j.1744-313X.2006.00582.xGoogle Scholar
- Carvelli J, Piperoglou C, Farnarier CC. Functional and genetic testing in adults with hlh do not reveal a cytotoxicity defect but rather an inflammatory profile. Blood. 2020; 136(5):542-552. https://doi.org/10.1182/blood.2019003664Google Scholar
- Mancebo E, Allende LM, Guzman M. Familial hemophagocytic lymphohistiocytosis in an adult patient homozygous for A91V in the perforin gene, with tuberculosis infection. Haematologica. 2006; 91(9):1257-1260. Google Scholar
- Fardet L, Galicier L, Lambotte O, Marzac C, Aumont C, Chahwan D. Development and validation of the HScore, a score for the diagnosis of reactive hemophagocytic syndrome. Arthritis Rheumatol. 2014; 66:2613-2620. https://doi.org/10.1002/art.38690Google Scholar
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