PMC5945832_01
CD40L expression in activated lymphocytes. Peripheral blood mononuclear cells from the patient and a healthy control were stimulated with ionomycin (1 mug/mL) and PMA (20 ng/mL) for 3 h, and non-adherent cells were then collected, stained and analyzed by flow cytometry. CD40L expression and CD40Ig binding were analyzed in CD3+CD4+ gated cells. (A) The patient exhibited reduced expression of CD40L after stimulation compared with that seen in the healthy control and displayed reduced CD40-Ig binding. (B) Representative histograms showing the reduction of CD40L expression and CD40Ig binding in the patient. The experiments were performed in duplicate and repeated twice with two distinct healthy controls.
fped-06-00130-g0001.jpg
PMC5945832_01_fped-06-00130-g0001.jpg
F1
Although the patient exhibited normal activation of CD3+CD4+ lymphocytes, as indicated by expression of the activation marker CD69 (data not shown), he showed a reduced frequency of CD40L expression after stimulation compared with that seen in the healthy control (23 and 65%, respectively; Figure 1A). In accordance with the reduced expression of CD40L, the binding of CD40-Ig to CD40L was reduced in the patient compared with that seen in the healthy control (14 and 34%, respectively; Figure 1A). Representative histograms are shown in Figure 1B.
Family pedigree and genetic analysis of the CD40L gene. Genetic analysis was performed on the patient and the patient's daughter and mother by Sanger sequencing. All five exons of CD40LG were analyzed. (A) Family pedigree (the patient is indicated by the arrow). (B) Chromatogram showing the patient's 6-nucleotide insertion in exon 1 and the heterozygous genotype of the patient's daughter, in contrast to the patient's mother and a healthy control. (C) Representation of the predicted insertion of 2-amino acids in the sequence of the mutated protein.
fped-06-00130-g0002.jpg
PMC5945832_01_fped-06-00130-g0002.jpg
F2
Genetic analysis performed through Sanger sequencing of the CD40LG revealed a 6-nucleotide insertion in exon 1 of CD40LG (c.121_122insCAGCAC), thus confirming the diagnosis of X-HIGM (Figure 2B). Taster predicted the addition of two amino acids (proline and alanine) to the protein sequence (Figure 2C). The patient's mutation was not found in any of the databases that were searched (dbSNP, ExAC, Genome Mutation, and 1,000 Genomes Project), which confirmed that it was a novel mutation. Because the mutation found in the patient was located on the X chromosome, genetic analysis was performed on the patient's daughter, and she was found to be heterozygous for the mutation, as expected (Figure 2B). To investigate the origin of the mutation, a genetic analysis was also performed on the patient's mother, which revealed that she did not carry the mutation found in the patient; thus, this mutation is a novel de novo mutation (Figure 2B). The family pedigree is presented in Figure 2A.