Supplementary methods
Molecular genetic analysis
Genomic DNA was extracted from peripheral blood leukocytes. All exons and flanking
introns of the ELANE gene were amplified by polymerase chain reaction (PCR) using
primer sets 1, 2 and 3 (Supplementary Table 1) and sequenced. Total RNA was
extracted from peripheral blood leukocytes. Complementary DNA (cDNA) was then
synthesized and subjected to PCR analysis using primer set 4 (Supplementary Table 1).
The PCR products were cloned into the pGEM-T Easy vector (Promega; Madison, WI,
USA), and individual alleles were analyzed by the restriction fragment length
polymorphism method using the BsiWI restriction site that specifically restricts the PCR
product corresponding to the wild-type (WT) allele. The full length WT ELANE gene was
amplified by PCR using primer set 5 (Supplementary Table 1). The PCR products were
inserted into the pGEM-T Easy vector and their sequences were confirmed. Constructs
encoding the G185R (SCN), L177F (CyN) and ΔV161–F170 mutant forms of ELANE
were generated by PCR-based mutagenesis of the WT construct using primer set 6
(Supplementary Table 1). The resulting fragments were inserted, in-frame, between the
BamHI and EcoRV sites of the pcDNA-C-Flag mammalian expression vector. Primer
sequences and PCR conditions are available on request.
Cell purification and massively parallel DNA sequencing (MPS) analysis of the ELANE
gene amplicons
Mononuclear cells and polymorphonuclear leukocytes were isolated from
peripheral blood by density gradient centrifugation using Lymphoprep (Fresenius Kabi
Norge AS, Oslo, Norway). CD3+ T cells or CD14+ monocytes from mononuclear cells
and CD16+ granulocytes from polymorphonuclear leukocytes were then purified by flow
cytometry (FACS Aria; BD Biosciences; Franklin Lakes, NJ, USA). To stain the cells,
the following antibodies were used: PE-conjugated anti-CD3 antibody, FITC-conjugated
anti-CD14 antibody and FITC-conjugated anti-CD16 antibody (BD Biosciences).
Genomic DNA was extracted from the buccal mucosa and the purified cells described
above using the QIAamp DNA Investigator Kit (Qiagen, Hilden, Germany). To fuse the
primer binding sequences for MPS, the genomic DNAs were used as templates in PCR
using primer set 7 (Supplementary Table 1), in which the specific primer sequences
were flanked by common 15-base adapter sequences (TGTAAAACGACGGCC and
GGAAACAGCTATGAC) at their 5′ ends. The PCR products were analyzed by MPS and
the statistical confidence of detected variations was estimated by the P-value as
previously described [11]. The mutation alleles analyzed, as shown in Table 1, had P-
values of less than 10−24 for each strand and the mutation frequencies did not show any
significant strand bias. This analysis was repeated using samples collected on a different
day and the data were consistent between experiments.
Quantitative reverse transcription-PCR
Human osteosarcoma (U2OS) cells were maintained in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum. Plasmids carrying the WT ELANE
allele and each of the mutant ELANE alleles were transfected into U2OS cells by
lipofection (Invitrogen, Carlsbad, CA, USA). After 24 hours, transfected U2OS cells were
subjected to quantitative PCR analysis. Total RNA was then extracted using an RNeasy
Mini Kit (Qiagen). The cDNA was synthesized directly with random primers, by reverse
transcription. BiP mRNA levels were determined by quantitative PCR, with the
StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and
the TaqMan Fast Advanced Master Mix Reagents Kit (Applied Biosystems). The results
were normalized with respect to the values obtained for endogenous GAPDH.
Supplementary Table 1. Primer sets used in this study
Primer set 1
Primer set 2
Primer set 3
Primer set 4
Primer set 5
Primer set 6
Primer set 7
F:
5′- CGAGCCAATCCAGCGTCTTGTC -3′
R:
5′- TGGCTTCACCGCTCAGAACCTC -3′
F:
5′- GATCCCGTGGGTTCCTGG -3′
R:
5′- TCAACGGCCCATGGCGGGTAT -3′
F:
5′- TGGAACCTGAGATGGGGAAACTG -3′
R:
5′- AGATGCTGGAGAGTGTGGGTGTG -3′
F:
5′- TGCAGGAGCTCAACGTGACG -3′
R:
5′- GGATGATAGAGTCGATCCAG -3′
F:
5′- GAATTCCACGGAGGGGCAGAGACCCCG -3′
R:
5′- GTCACCTCAGCTGCCCAC -3′
F:
5′- GTCTGCACTCTCGGGGACTCCGGC -3′
R:
5′- GCCGGAGTCCCCGAGAGTGCAGAC -3′
F:
5′- GGATGATAGAGTCGATCCAG -3′
R:
5′- GTGGATTAGCCCGTTGCAG -3′