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. 2001 Oct 23;98(22):12614-9.
doi: 10.1073/pnas.221456898.

Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM

S Ferrari  1 , S Giliani, A Insalaco, A Al-Ghonaium, A R Soresina, M Loubser, M A Avanzini, M Marconi, R Badolato, A G Ugazio, Y Levy, N Catalan, A Durandy, A Tbakhi, L D Notarangelo, A Plebani
Affiliations

Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM

S Ferrari et al. Proc Natl Acad Sci U S A. .

Abstract

CD40 is a member of the tumor necrosis factor receptor superfamily, expressed on a wide range of cell types including B cells, macrophages, and dendritic cells. CD40 is the receptor for CD40 ligand (CD40L), a molecule predominantly expressed by activated CD4(+) T cells. CD40/CD40L interaction induces the formation of memory B lymphocytes and promotes Ig isotype switching, as demonstrated in mice knocked-out for either CD40L or CD40 gene, and in patients with X-linked hyper IgM syndrome, a disease caused by CD40L/TNFSF5 gene mutations. In the present study, we have identified three patients with an autosomal recessive form of hyper IgM who fail to express CD40 on the cell surface. Sequence analysis of CD40 genomic DNA showed that one patient carried a homozygous silent mutation at the fifth base pair position of exon 5, involving an exonic splicing enhancer and leading to exon skipping and premature termination; the other two patients showed a homozygous point mutation in exon 3, resulting in a cysteine to arginine substitution. These findings show that mutations of the CD40 gene cause an autosomal recessive form of hyper IgM, which is immunologically and clinically undistinguishable from the X-linked form.

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Figures

Figure 1
Figure 1
CD40 is absent from the surface of B cells. Peripheral blood B cells from Pt.1 and Pt.2 and from a normal control were analyzed by flow cytometry for surface expression of CD40. PBMC were double stained for FITC anti-CD40 and PE anti-CD19 and gated on the CD19+ population. Staining with mouse IgG1-FITC represented the isotype-matched internal control.
Figure 2
Figure 2
Western blot analysis of CD40 expression in HIGM3 patients. Western blot experiment was performed with whole cellular extracts from B-LCLs derived from the patients and from a normal control (Ctl). Probing with an anti-CD40 polyclonal antibody, directed against the amino terminus of the protein, displays the expected 40-kDa band in the control and a lower molecular weight band in Pt.2. Both bands are present in patient's mother (Pt.2 Mo). No CD40-specific signal is detectable in Pt.1, even after prolonged exposure of the filter. (Lower) The same filter reblotted with an anti-STAT5 antibody as a control.
Figure 3
Figure 3
RT-PCR of CD40 mRNA from PBMCs of control, Pt.1, and Pt.2, and the mother of Pt.1 (Pt.1 Mo). The normal amplification product obtained with primers P1 and P2 is 550 bp long. The lower band (Pt.1 and Pt.1 Mo) contains a deletion of 94 bp corresponding to exon 5 (Δ5 in the figure). Both amplification products were cloned and sequenced. Also indicated are the position of additional primers used in this work and the stop codon created by skipping of exon 5. (Lower) A control RT-PCR reaction performed with β-actin specific primers.
Figure 4
Figure 4
Genomic organization of the CD40 gene, showing the positions of the point mutations and the sequence data (sense strand) of the relevant regions (exon 3, nucleotides 290–296, and exon 5, nucleotides 451–458, respectively). In Pt.2 and Pt.3, the T to C substitution at nucleotide 294 changes codon 83 from Cys to Arg. In Pt.1, the A to T substitution at nucleotide 455 is a silent mutation that occurs within a putative binding motif for the SF2/ASF protein. Base pair numbers are designated according to CD40 mRNA sequence (accession no. NM 001250). Also indicated are the scores for the normal and mutant exon 5 sequences obtained after calculating the frequency matrices derived from a pool of functional enhancer sequences selected in vitro (25). A score above 1.956 indicates a bona fide SF2/ASF binding motif (the consensus motif is indicated: S = G or C, R = A or G).
Figure 5
Figure 5
Exon trapping experiment from the human CD40 genomic clones. (A) Schematically shows the potential splicing events generated in the exon trapping procedure. COS-7 cells were transfected with the pSPL3 vector containing the CD40 genomic sequence corresponding to either the wild-type (WT) or the mutated (MUT) exon 5 and the flanking introns, or with the empty plasmid (EP). RNAs from transfected COS-7 cells were isolated, converted to cDNA, and amplified (primers used in the secondary amplification reaction are indicated). The resulting PCR products were analyzed by 2% agarose gel electrophoresis (B), subcloned, and sequenced. The 177-bp fragment was derived from the splicing occurring between vector 5′ and 3′ splice sites (lane 1). This product was absent when the cells were transfected with the construct containing the wild-type CD40 exon 5 sequence (lane 2), but it reappeared when the CD40 exon 5 carried the A455T mutation present in Pt.1 (compare lane 2 and 3). The 271-bp specific fragment present in lane 2 corresponded to the product derived from the splicing occurring between the vector and the CD40 exon 5, and it included the exon 5. A weakly stained 300-bp fragment was present in lanes 1 and 3 (CS in the figure) and corresponded to a product derived from cryptic splice sites, which are present into the intron of the HIV 1 tat gene. Southern blot analysis of the PCR products, performed with a probe corresponding to the CD40 exon 5, confirmed that only the specific 271-bp fragment in lane 2 trapped the exon 5 (lane 5).
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