*2.5. Next-Generation Sequencing (NGS) and Sanger Validation*

EDTA blood samples were subjected to an automated extraction and purification process to obtain genomic DNA using the QIAsymphony SP (Qiagen, Hilden, Germany). The proband sample was prepared for an NGS study. We performed targeted sequencing using a designed panel of 41 genes associated with primary antibody immunodeficiency (*AICDA, ATP6AP1, BLNK, BTK, CARD11, CD19, CD40, CD40LG, CD79A, CD79B, CD81, CR2, CTLA4, CXCR4, ICOS, IGLL1, IKZF1, IL21, INO80, IRF2BP2, LRBA, LRRC8A, MOGS, MS4A1, MSH6, NFKB1, NFKB2, PIK3CD, PIK3R1, PLCG2, PMS2, PTEN, SEC61A1, TCF3, TNFRSF13B, TNFRSF13C, TNFSF12, TNFSF13, TRNT1, TTC37, UNG*). The library was created using the SureSelect XT Reagent library preparation kit (Agilent Technologies, Inc., Santa Clara, CA, USA) for paired-end multiplexed sequencing of Illumina (Inc., San Diego, CA, USA). Target regions were enriched with the Custom SureSelect probe kit (Agilent). Cluster preparation was performed using the cBot device, and library sequencing was performed using the Illumina HiSeq1500 platform (Illumina, Inc., San Diego, CA, USA). Bioinformatic analysis was applied through an end-to-end in-house pipeline developed by Health in Code (A Coruña, Spain), in accordance with the best WES analysis practices. The identified pathogenic variant in the proband was confirmed by Sanger sequencing, by sequencing exon 5 of the *IKZF1* gene bidirectionally, with its intronic flanking regions. Sanger sequencing was also performed in the patient's parents.

#### *2.6. SARS-CoV-2 Serological Study*

In the context of the SARS-CoV-2 pandemic, the patient received the complete vaccination program against COVID-19: the first and second dose with the vector SARS-CoV-2 vaccine (Oxford-AstraZeneca AZD1222, ChAdOx1 nCoV-19, Vaxzevria) and the third dose (booster dose) with the messenger RNA SARS-CoV-2 vaccine (mRNA-1273, Moderna).

We analyzed the humoral immune response against the SARS-CoV-2 Spike (S) protein one and three months after the second dose with AstraZeneca and one month after the third dose with Moderna to ensure complete vaccination and subsequent booster. Quantitative determination of IgG against protein S was performed using the chemiluminescent microparticle immunoassay (CMIA) in the Alinity autoanalyzer (Abbott) following the manufacturer's instructions with the SARS-CoV-2 IgG II Quant Assay kit. Results were expressed in binding antibody units per milliliter (BAU/mL). A test was determined as positive if the signal was >7.5 BAU/mL.
