HUMAN MUTATION 3:73-75 (1994)

MUTATION IN BRIEF

C to T Mutation Causing Premature Termination of CD40 Ligand at Amino Acid 221 in a Patient Affected by Hyper IgM Syndrome A. Villa*, D. Strina, P. Macchi, M.C. Patrosso, P. Vezzoni, P.A. TOVO, S . Giliani, A.G. Ugazio, and L.D. Notarangelo

Istituto di Tecnologie Biomediche Awanzate, CNR, Milano (A. V., D. S., P. M., M. C. P., P. V.); Clinica Pediatrica e Istituto di Chimica, Uniwersitd di Bresciu, Brescia (S. G., A. G. U., L. D.N.); Clinica Pediatrica, Uniwersitd di Turino, Torino, Italy (P.A. T.); Fax: 392-266-3030

Communicated by Sergio Ottoknghi

The X-linked form of Hyper IgM syndrome is a rare disorder characterized by the inability of B cells to undergo isotype switch. Affected individ- uals present normal or high serum concentrations of IgM and markedly decreased levels of IgG, IgE, and IgA (Notarangelo et al., 1992). The disorder is characterized by severe bacterial and opportunis- tic infections, increased susceptibility to neo- plasms, and autoimmunity. The patient’s quality of life is poor, despite regular administration of intra- venous immunoglobulins (Ig).

Recently, the gene responsible for this disease and its mouse homologue have been cloned and characterized by various groups and have been shown to be the ligand for CD40 antigen (CD40L). The encoded product is a transmem- brane protein 261 AA long, expressed in activated T cells that interact with the B cell surface mole- cule CD40 (Armitage et al., 1992; Graf et al., 1992; Hollenbaugh et al., 1992; Spriggs et al., 1992). Because of homology with the tumor ne- crosis factor (TNF)-a, the CD40L has also been designated TRAP (TNF-related activated protein, Graf et al., 1992). Upon interaction with CD40 and proper costimulation with cytokines (IL-4, IL- 10) CD40L induces Ig switch and B cell prolifera- tion (Spriggs et al., 1992). Its expression appears to be tightly regulated since strong expression can be induced 2-12 hr after activation with both Ca2+ ionophore A23187 and PMA but not with only one of them (Graf et al., 1992).

The predicted protein structure shows an intra- cellular domain at the NH2 terminus (AA 1-22) followed by a transmembrane domain (AA 23-46) and an extracellular portion at the COOH termi-

0 1994 WILEY-LISS, INC.

nus (AA 47-261). By comparison with TNF-a, putative sites for trimer formation have been iden- tified at positions 118-121, 174, 214-215, and 25 1-252, while AA involved in receptor binding are clustered between positions 121 and 169 (Di- Santo et al., 1993). All these putative active sites are contained in the extracellular domain.

Mutations of this gene at the molecular level have been demonstrated by various groups in a total of 12 cases (Aruffo et al., 1993; DiSanto et al., 1993; Korthauer et al., 1993; Allen et al., 1993). Among these, 11 have been detected in the extracellular domain, with the 12th being located in the transmembrane domain. Among the muta- tions in the extracellular domain, one is a 63 bp in-frame deletion (AA 116-136), two are smaller out-of-frame deletions (8 and 10 bp deletions start- ing at AA 136) causing truncation of the transla- tion product (DiSanto et al., 1993), while the oth- ers are point mutations affecting AA 123, 128, or 129 (these two changes are present in the same patient), 140 (two different mutations in the same codon, one creating a stop codon), 155, 211, 227, and 235. Among the single amino acid substitu- tions, only the one at AA 123 involves one of the putative trimerization or receptor binding sites. The transmembrane abnormality is a single point mutation at AA 36 (Korthauer et al., 1993). More recently two other incompletely characterized mu- tations have been reported (Fuleihan et al., 1993).

We describe here a new mutation at the DNA

Received May 7, 1993 accepted June 16, 1993. *To whom reprint requestskorrespondence should be ad-

dressed.

74 VILLAETAL.

FIGURE 1. Nucleotide sequence of region around nucleotide 682 of CD40L gene horn a normal control and DL patient. A “C to T” transition (arrow) is shown.

level in a patient affected by Hyper IgM. This pa- tient (D.DL.) is a 6-year-old male child, born from nonconsanguineous parents. A brother of the ma- ternal grandmother had died of recurrent infec- tions at 4 years of age. The proband presented at 18 months of age with recurrent respiratory tract infections. Hypogammaglobulinemia with normal levels of IgM was documented (IgG 142 mg/dl; IgM 138 mg/dl). Since then, low serum Ig levels (100-200 mg/dl), undetectable IgA and increased IgM (up to 378 mgidl) have been recorded. Immu- nodeficiency with Hyper IgM was diagnosed and treatment with intravenous Ig (IVIG) was started in November 1990. Since April 1992, the child presented cyclic neutropenia and recurrent stoma- titis, a well known complication of Hyper IgM syn- drome. Upon treatment with IVIG, serum IgM levels have decreased to 150-200 mg/dl.

All the previously reported mutations have been detected by reverse PCR on activated peripheral blood lymphocytes. Unfortunately, fresh cells were not available for stimulation and RNA isolation, and our analysis had to rely on DNA. Since the genomic structure of CD40L is not yet known, we tried several pairs of primers and were able to am- plify most of the extracellular region with the fol- lowing primers:

p3 79F: CCTCAAATTGCGGCACAT (sense primer starting at nucleotide 379 of the 215017 sequence in data bases) p483R: TGTTTCCCATTTTCCAGG (antisense primer starting at nucleotide 483) p483F: CCTGGAAAATGGGAAACA (sense primer starting at nucleotide 483) p82 1R: AGCTCCACCACAGCCTGC (an- tisense primer starting at position 821).

These two primer pairs give the nucleotide se- quence of the CD40L portion between AA 122 and 261, a region where 11 out of the 12 mutations described so far are contained. Amplification prod- ucts were cloned in TA cloning vector and se- quenced with Sanger technique. A “C to T” tran- sition at nucleotide 682 (with reference to the sequence Z150 17) creating a stop codon (TAA) at codon 221 was detected. To avoid the possibility of PCR artifacts, two independent clones were se- quenced. This mutation truncates the protein by eliminating the last 40 AA. No restriction site was disrupted or created by this mutation. Since DNA from other affected or carrier relatives was not available, we were not able to formally prove that this mutation was derived from the mother, as sug- gested by the clinical report of an affected grand- mother’s brother.

All the mutations detected so far affect protein structure differently. Previously reported trunca- tion products eliminate about half of the protein including most of the trimerization sites. The trun- cated protein reported here lacks only the last 40 AA leaving all the receptor binding sites intact. The inability to bind CD40 can be explained by the elimination of the last putative trimerization site at AA 251-252. However, it must be pointed out that gp39 protein structure appears to be very sensitive to single substitutions even if they do not involve putative binding or trimerization sites, as demonstrated by the fact that most reported single AA changes do not involve predicted binding or trimerization sites.

ACKNOWLEDGMENTS

This work is supported in part by grants from CNR, PF Ingegneria Genetica Sottoprogetto 5 (to P.V.) and Sottoprogetto 4 (to A.U.), Biotecnolo- gie e Biostrumentazione (to C.P.), ACRO (to A.V.), and from Telethon (to P.V. and A.U.). We would like to thank Mr. Arrigo Caraffini for oligonucleotide preparation and Mrs. L. Susani and Mr. M. Littardi for technical assistance.

PREMATURE TERMINATION OF CD4OL IN HYPER IgM SYNDROME 75

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