Male patient, product of the first pregnancy, with prenatal controls, good evolution and spontaneous vaginal delivery.
You have a complete vaccination course without abnormal reactions.
At two months of age she had acute diarrheal disease and recurrent perianal abscesses.
At 6 months of age she presented chronic inflammation of the colon and bacterial colitis caused by Salmonella sp, required hospitalization for 3 weeks with antibiotic treatment.
At 3 years of age she consulted again because of flu-like symptoms, lower respiratory tract infection, erythema in the perianal region, dyspeptic symptoms and lymphadenopathy in the posterior cervical chain.
A year later, he began to present with fungal infection of the face, diffuse abdominal pain, occasional intestinal habit three to four times a day and abscess in the perianal region.
Within the family history, the mother reported recurrent abscesses since she was 17 years old but did not receive medical treatment.
Nitroblue Tetrazolium (NBT) Reduction
37 patients, their mothers and healthy controls were taken peripheral blood samples without anticoagulant with a 1 ml syringe and 6 were deposited on a pre-warmed portal vein 37C optically washed with 1 ml paste finally formed.
Two hundred neutrophils were counted in the immersion field (100X), the generation of oxygen radicals was evidenced by the reduction of NBT dye in cells activated with PMA blue, with the consequent formation of dark blue precipitates.
More than 95% of normal neutrophils should reduce NBT.
Cit paste flow with dihydrorodamine (DHR)
4 ml of peripheral venous blood was taken with EDTA from the patient, his mother and the healthy control.
400 μl of blood plus 4 ml of a lysis (Clor of Arsenic 8.3 mg/ml, sodium 0.84 mg/ml and EDTA 0.5 M) were centrifuged at 5 g later 37° bicarbonate solution.
The cell button obtained was washed with HBSS buffer (Hank's Buffered Salt Solution), 400 μL of a working solution was added to a non-hydrogen phosphate buffering system HBSS at 37 μC
Flow cytometry was performed on a Becton Dickinson FACSC II cytometer.
Separation of peripheral blood mononuclear cells (PBMC)
We took 15 ml of peripheral venous blood in tube with heparin from the patient, his mother and healthy control.
The sample was diluted in PBS buffer (proportion 1:2), this mixture was deposited in a tube containing Ficoll 1077 in a proportion 1:3 with respect to the mixture temperature and centrifuged at room temperature g mononuclear min.
Purity and viability of the cells were determined by counting in a Neubauer chamber using gentian tapering and trypan blue staining, respectively.
The isolated cells had viability and purity percentages above 95%.
RNA extraction, cDNA synthesis and PCR
RNA was extracted from PBMC by TRIzol method (Invitrogen).
Copy DNA (cDNA) was obtained from total RNA by reverse transcription using ThermoScript RT-PCR System (Invitrogen) according to manufacturer specifications.
For the study of cDNA of gp91 phox, a strategy was used in which three overlapping regions were amplified in order to cover the entire exome, for this purpose three pairs of primers 7-13 were used.
1.
PCR conditions were: 2 min at 95°C, 30 cycles that included 95°C for 15 s, 63°C for 30 s (regions 1-5 and 7-13) and 72°C saphenous agar for 30 s.
Fragments were 450 bp for region 1-5, 700 bp for region 3-9 and 900 bp for region 7-13.
Genomic DNA extraction and PCR
Genomic DNA (gDNA) was extracted from PBMC by the TRIzol method (invitrogen) according to the manufacturer's specifications.
Using specific primers or primers, exon 2 of the CYBB gene was amplified including the edges of introns 1 and 2.
PCR conditions were 2 min at 95°C, 30 cycles including 95°C for 15 s, 63°C for 30 s and 72°C for 45 s, and a final extension at 72°C.
The fragment obtained was approximately 230 bp.
Simple chain conformation polymorphisms (SSCP)
The conformal polymorphism of fragments generated by PCR of genomic DNA was studied.
The amplified products plus load buffer (5% bromphenol blue, 0.05% xylene oxide, 95% formamide and 20 mM EDTA) were immediately placed at 95°C for 5 min.
Samples were run by a non-denaturing electrophoresis (non-denaturing a 12% acrylamide gel dissolved in buffer TBE 10% glycerol) using 90 volts 10 h at 10 h.
The gel with the AND products was fixed with 10% acetic acid for 30 min and stained with 0.15% sodium nitrate Candide and 0.14% formaldehyde solution for 30 min.
The migration profile of the patient and mother bands was compared with the healthy control.
Sequencing
The products obtained by cDNA and gDNA PCR were sequenced with standard techniques (Macrogen Inc. Seul, Korea).
Sequences of patients and controls were compared with normal sequences from GeneBank data (Núcure access NM_000397).
This study was approved by the Ethics Committee of the Faculty of Medicine of the University of Antioquia.
Medellin, Colombia.
The identity of the subjects in this study remains confidential.
