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].