Patients and methods:
This study was carried out in the Allergy-Immunology Unit, Advanced Paediatrics Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, North India. Our institute is a federally funded, not-for-profit tertiary care referral hospital for North-West India. The study included all patients diagnosed to have HAE between January 1996 and December 2019. Data were retrieved from medical records and entered in a predesigned proforma. The study was approved by Institute Ethics Committee and an informed consent was obtained from patients or their parents. Serum complements C4 was measured by end-point nephelometry using a semi-automated nephelometer, MININeph from The Binding Site, Birmingham, UK. Functional C1-INH activity was assessed by MicroVue C1 inhibitor Plus enzyme immunoassay kit from Quidel, USA. Our laboratory is regularly participating in an external quality assurance scheme i.e. UK National Quality Assurance Scheme (UK NQAS) for special proteins since 2010. Quantitative C1-INH was estimated using radial immunodiffusion method and by semi-automated nephelometry. Patients were diagnosed to have HAE if they had characteristic clinical manifestations of disease with or without a suggestive family history with low C4 and either a low quantitative C1-INH (type 1 HAE) or low functional activity of C1-INH (type 2 HAE). Patients with suspected HAE but with normal quantitative or functional C1INH levels were not included in this analysis. Family members of patients with HAE for whom complete clinical details and laboratory data were not available were also excluded from this analysis.
Because of non-availability of recombinant or plasma derived C1-INH therapy in India, stanozolol 2-4 mg/day), danazol (100-600 mg/day) or tranexamic acid [TA] (30-50 mg/kg/day) was used for long-term prophylaxis. For short term prophylaxis during planned surgeries, stanozolol (2-4 mg/day) and fresh frozen plasma, FFP (10 ml/kg) were used. Acute episodes of life-threatening laryngeal oedema were managed using FFP (10 ml/kg).
Sanger sequencing for SERPING1 gene:
We started doing Sanger sequencing of the SERPING1 gene in our cohort of patients with HAE since 2018. For Sanger sequencing, 200 µl of EDTA blood was used. Genomic DNA was extracted using QIAamp DNA extraction kit as per the manufacturers’ protocol (Qiagen, Hilden Germany). All exons of SERPING1 gene were amplified using oligonucleotide primers as mentioned in Table 1 and Figure 1. These oligonucleotide primers were designed to cover exon/intron junctions of all exons. Each exon was amplified using Polymerase Chain Reaction (PCR). The PCR product was checked on 1.5% of agarose gel electrophoresis followed by purification and this purified product was used for Sanger Sequencing using the ABI Big Dye terminator kit and Agilent 2100 Bioanalyzer System. The sequencing data were analyzed using Finch TV and Codon code aligner software. In silico prediction analysis was used for all novel mutations detected in theSERPING1 gene. The pathogenic nature of these variants was inferred using 3 free, online bioinformatics tools for prediction of functional effects of amino acid substitution in proteins viz. Provean, PolyPhen-2 and FATTHM.