Taqman snp genotyping allelic discrimination method

Genotyping for rs12979860 and rs8099917 SNPs was undertaken using the TaqMan SNP genotyping allelic discrimination method (Applied Biosystems, Foster City, CA, USA). The rs8099917 genotyping kit was supplied by Applied Biosystems and rs12979860 genotyping was performed using a custom-designed genotyping assay from Applied Biosystems. Genotyping was performed using the StepOne RT system and analysed with StepOne software v.2.3.0 (Applied Biosystems). All genotyping was undertaken blinded to clinical variables.

Fasting plasma glucose, total cholesterol, HDL-C, triglyceride, uric acid, and creatinine levels were measured using standard clinical chemistry methods (Glucosio HK UV; Colesterolo totale Mod P/D; Colesterolo HDL gen 3 mod P/917; Trigliceridi; Acido urico MOD P/917; Creatinina enzimatica; Roche Diagnostics, Monza, Italy). Basal insulin concentrations (Elecsys insulina; Roche Diagnostics; Monza, Italy), high-sensitivity C-reactive protein (hs-CRP; B-analyst hs-CRP; Menarini Diagnostics; Florence, Italy), and glycated hemoglobin (HbA₁c; B-analyst HbA1c; Menarini Diagnostics; Florence, Italy) levels were also measured. Low-density lipoprotein-cholesterol serum concentration was calculated using Friedewald’s formula [20 (link)]; glomerular filtration rate (eGFR) was estimated based on the CKD-EPI equation [21 (link)], and HOMA-IR was calculated as described by Matthews et al. [22 (link)]. DNA was purified using a QIAmp blood Mini Kit (Qiagen, Hilden, Germany), and DNA samples were quantified using spectrophotometry. Genotyping for patatin-like phospholipase domain containing 3 (PNPLA3) (rs738409) and transmembrane 6 superfamily 2 (TM6SF2) (rs58542926) was performed using the TaqMan SNP genotyping allelic discrimination method (Applied Biosystems, Foster City, CA, USA).

After obtaining written informed consent from at least one parent, a blood sample was collected from 79 hospitalized infected children, who consented also to genetic analysis, in order to evaluate the contribution of genetic factors to the clinical variability of COVID-19. DNA was extracted from peripheral blood by the QIASymphony (Qiagen, Milan, Italy). Genotyping for common genetic risk factors for COVID-19 susceptibility or severity in adult patients (LZTFL1 rs11385942 tagging chromosome 3 cluster, OAS3 rs10735079 tagging the OAS1/2/3 locus variability, FUT2 rs601338, IFNAR2 rs2229207, and DPP9 rs2109069) was carried out using the TaqMan SNP genotyping allelic discrimination method (Applied Biosystems). Commercial genotyping assays were available. Genotypes were analyzed by the SDS software v.2.3 (StepOne Plus; Applied Biosystems). ABO genetic locus variation was determined using the rs657152, as previously described (14 (link)–20 (link)). The frequencies of these variants observed in children were compared to that of a control cohort (2,848 healthy subjects) and an adult cohort with severe COVID-19 (995 patients) (23 (link)). The genetic study was approved by the Ethics Committee of the Fondazione IRCCS Ca’ Granda (FoGS, approval number 342_2020).

Genotyping for IFNL3 rs12979860 and rs8099917 and for PNPLA3 rs738409 was carried out using the TaqMan SNP genotyping allelic discrimination method (Applied Biosystems, Foster City, CA, USA). The genotyping was performed with SDS software v.1.3.0 (ABI Prism 7500, Foster City, CA, USA).

Genotyping for MICA rs2596542 and DEPDC5 rs1012068 was contracted to the Australian Genome Research Facility (AGRF; QLD, Australia). 1051 samples were genotyped using the Sequenom MassARRAY system and iPLEX Gold chemistry while 638 samples were genotyped using the TaqMan SNP genotyping allelic discrimination method (Applied Biosystems, Foster City, CA, USA). All genotyping was blinded to clinical variables.

DNA was purified using the QIAmp blood Mini Kit (Qiagen, Mainz,Germany) and DNA samples were quantified using spectrophotometric determination. Genotyping for MERTK (rs4374383 A>G SNP and rs6726639 A>C SNP) was carried out using the TaqMan SNP genotyping allelic discrimination method (Applied Biosystems, Foster City, CA, USA). Genotyping assays were commercially available. Genotypes were called using Sequence Detection Software (SDS v.2.3) (Applied Biosystems, USA). Genotyping was conducted in a blinded fashion relative to patient characteristics.