2.3 SERPING1 gene mutational screening
DNA was extracted from peripheral white blood cells using standard protocols. To identify Single Nucleotide Variants (SNV), exonic regions of SERPING1 were amplified from samples of total genomic DNA. Primary mutational screening was performed by Single Strand Conformational Polymorphism (SSCP). DNA fragments with abnormal mobility compared to wild type control were subsequently analysed by Sanger sequencing. Regarding Copy Number Variation (CNV) analysis ofSERPING1 , we used Multiplex Ligation-dependent Probe Amplification (MLPA) technique. Capillary electrophoresis was performed on ABI 3130 Genetic Analyzer, (Applied Biosystem, USA).
We also used custom Next Generation Sequencing (NGS) Panel (Illumina, USA) of 347 genes most frequently mutated in patients with Primary Immune Deficiency (PID) and HAE. Sequencing was performed on MiSeq platform (Illumina, USA). Observed variants were confirmed by Sanger resequencing. Data interpretation included population filtering, functional prediction (SIFT, PolyPhen2, Hope) and other necessary steps according to Joint Recommendation of the Association for Molecular Pathology and the College of American Pathologists [28].