These are caused by mutations in any of the five subunits that le

These are caused by mutations in any of the five subunits that leave the protein expression intact but destroy the enzymatic activity

of the assembled oxidase complex. In that case, direct sequencing of all five genes can be considered. Alternatively, a cell-free oxidase assay may be used to distinguish a defect in a cytosolic component (p40phox, p47phox or p67phox) from a defect in a membrane-bound component (gp91phox or p22phox). For this assay, neutrophil membranes from the patient are mixed with neutrophil cytosol from a healthy donor (or vice versa), incubated Dinaciclib price with NADPH and γS-GTP, and activated with an amphiphilic agent [low concentrations of sodium dodecyl sulphate (SDS) or arachidonic acid] [27]. The resulting oxidase activity can be measured by superoxide formation or oxygen consumption and is used to localize the defect to either the cytosol or the membrane fraction. Identification of the mutated gene that causes the defect in NADPH oxidase activity can also be made if transfection of the patient’s Epstein–Barr virus (EBV)-transformed B lymphocytes with retroviral vectors that contain the wild-type cDNA restores this activity [28]. For a detailed protocol, see [27]. For a protocol, see [28]. The disease-causing mutation should be determined in every CGD patient. This is necessary

for undisputable proof of which gene is affected, and as such the basis for genetic counselling. Ferroptosis inhibitor clinical trial Carriers of the disease without clinical symptoms can only be diagnosed reliably by mutation analysis. Also, in case prenatal diagnosis or gene therapy is an option in the family, this information must be available. When patients are transplanted with stem cells from a family member, it is important to know that this donor is not carrying the mutation. Finally, this information helps investigators to link medical expression of CGD to the genetic cause. Genomic DNA and RNA can be extracted from the mononuclear leucocyte fraction [peripheral blood mononuclear cells (PBMC)] obtained as a side product during neutrophil Endonuclease purification [12]. The CYBB, CYBA, NCF2 and NCF4 genes (for

properties see Table 1) can be analysed from genomic DNA by polymerase chain reaction (PCR) amplification and sequencing. NCF1 is more difficult, because it is accompanied on each side by one pseudo-NCF1 gene. These pseudo-NCF1 genes are >99% homologous to NCF1 but lack a GT sequence at the start of exon 2, which induces a frame-shift and a premature termination of protein synthesis. Therefore, NCF1-specific PCR is difficult, because the primers have to contain NCF1-specific sequences at the segregating points between NCF1 and its pseudogenes. It is recommended, therefore, to first perform a gene scan [29] to determine whether only GT-deletion-containing pseudogenes are present or whether one or two NCF1 genes are present in the patient’s DNA.

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