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.