2.1. Subject Enrollment

We obtained peripheral blood samples from 92 Spanish patients from Majorca Island with a confirmed SARS-CoV-2 viral RNA polymerase-chain-reaction (PCR) test from nasopharyngeal swabs. All enrolled individuals were infected during the first wave and were not vaccinated with SARS-CoV-2. The study was conducted according to the ethical guidelines of the 1975 Declaration of Helsinki and approved by the Balearic Islands Clinical Research Ethics Committee. Written informed consent to have their clinical and genetic information published in medical or scientific journals was obtained from all subjects.

With the objective of finding genetic risk or protective factors related to hyperinflammatory COVID-19, two cohorts with extreme phenotypes of SARS-CoV-2 infection were defined: (1) a hyperinflammatory patients (H-P) cohort and (2) asymptomatic or mild patients (AM-Risk-P) with a high risk (due to older age) of severe COVID-19 as a control cohort (Figure 1a).

Experimental methodology. (a) Cohort of patients: (1) the hyperinflammatory patient cohort H-P included 52 patients that showed hyperinflammatory characteristics according to a score for the diagnosis of reactive hemophagocytic syndrome (HScore) over 100; (2) the asymptomatic or mild patients cohort (AM-Risk-P) included 40 individuals at high risk (based on age greater than 70 years) for severe COVID-19 who remained asymptomatic or developed mild disease that we used as control group. (b) Multi-gene panel covering 24 genes related to different PIDs that predispose to cytokine storm distributed in two groups: autoinflammatory disorders or defects in immunity with predisposition to viral infection and diseases of immune dysregulation.

The H-P cohort included 52 patients with severe/critical COVID-19 admitted to Son Espases or Son Llatzer Hospitals (Table S1). The severity of signs and symptoms developed during hospitalization was categorized as severe (grade 2) or critical (grade 3). Severe disease was established when dyspnea was associated with a ≥30/min respiratory rate or <93% blood oxygen saturation or <300 partial pressure of arterial oxygen to the fraction of inspired oxygen ratio and/or >50% lung infiltrates within 24 to 48 hours from admission; critical disease was established for cases with respiratory failure, septic shock, and/or multiple organ dysfunction or failure. In addition, severe or critical patients were classified as hyperinflammatory based on a score for the diagnosis of reactive hemophagocytic syndrome (HScore) [19] over 100. None of the subjects had features of PIDs before the hyperinflammatory COVID-19 diagnosis.

Serum levels of IL-6, IL-10, IL-1, IL-1Ra, IL-18, and sIL-2Rα (sCD25) were analyzed in 22 H-P patients during the acute phase of COVID-19 (Table S1).

Forty individuals were included in the AM-Risk-P control cohort that was established as a risk group for developing severe disease (on the basis of age over 70) with patients who were asymptomatic or developed mild disease (Table S2).

All patients were Caucasian with Spanish ancestry, with the exception of three Hispanic patients.