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The candidate gene for this disease is the CD40 ligand, which is localized on the long arm of the X chromosome3 in the region to which the inherited form of the syndrome has been mapped4,5. The CD40 ligand is expressed on the surface of activated T cells and forms a complex with the CD40 receptor on B cells3,6,7,8. This interaction can induce B-cell proliferation and, in the presence of the appropriate cytokines, can promote switching of immunoglobulin isotypes9. Infants with the hyper-IgM syndrome have heterogeneous point mutations or deletions throughout the coding region of the CD40 ligand10,11,12,13. These genetic alterations result in the absence of CD40-binding activity. Abnormal interactions between the CD40 receptor and the CD40 ligand provide a molecular basis for the immunologic findings in the X-linked form of the hyper-IgM syndrome.
Genetic counseling and the ability to diagnose this disease prenatally have been limited by a number of factors, including the absence of immunologic abnormalities detectable during fetal and early postnatal life and the random pattern of X inactivation displayed by lymphoid cells from obligate carriers14. In addition, only a limited number of polymorphic DNA probes for linkage analysis have been characterized in the affected region of the X chromosome (Xq24-27). The identification of the CD40 ligand as the candidate gene for X-linked hyper-IgM immunodeficiency might lead one to envision a strategy in which affected families could be screened for clustered mutations. In a recent sequence analysis of 13 patients with X-linked hyper-IgM syndrome, however, 12 distinct point mutations or deletions in the CD40 ligand gene were described10,11,12,13. Thus, the marked heterogeneity of CD40 ligand mutations necessitates the sequencing of complementary DNA (cDNA) to identify affected persons -- a technique that may prove difficult to implement as a general method of screening.
In this report we characterize a highly polymorphic dinucleotide repeat in the CD40 ligand gene that we have used to diagnose X-linked hyper-IgM immunodeficiency prenatally during the first trimester of pregnancy. This intragenic polymorphic marker provides an easy-to-use method of diagnosis to aid in genetic counseling with regard to this disease.
Methods
Families with the Hyper-IgM Syndrome
Cases of X-linked hyper-IgM immunodeficiency were diagnosed in four families by clinical and immunologic investigations at the Hopital Necker-Enfants Malades and were classified according to the criteria of the World Health Organization Committee on Immunodeficiency15. Additional clinical information on deceased members of some kindreds was obtained from medical records. Peripheral-blood samples were obtained after the subjects had given written informed consent, and genomic DNA was prepared as described elsewhere16.
Prenatal Analysis
Chorionic-villus sampling was performed by transabdominal biopsy at 12 weeks of gestation. Karyotypic analysis was performed according to standard methods,17 and genomic DNA was isolated as described elsewhere16.
Microsatellite Typing
Genomic DNA samples from 36 healthy, unrelated women were used to determine the frequency of heterozygosity18 of the CD40 ligand dinucleotide repeat. Sequences of forward 5'CTTCTCAATCCCCTTTC3' and reverse 5'CATCCCTACTCCTCACC3' primers were chosen that flank the cytosine and adenine (CA) repeat of the CD40 ligand cDNA. The polymerase chain reaction (PCR) was carried out in a volume of 25 microl with 100 ng of genomic DNA, 3 pmol of the forward primer (end-labeled with [32P]gamma-ATP), 3 pmol of unlabeled forward primer, 6 pmol of unlabeled reverse primer, and 0.5 U of Taq polymerase in a buffer containing 0.4 mM deoxynucleotide triphosphates, 1.5 mM magnesium chloride, 50 mM potassium chloride, 10 mM Tris-hydrochloric acid (pH 9), and 0.1 percent Triton X-100. After an initial cycle of denaturation at 94 °C for five minutes, 27 cycles of denaturation at 94 °C for one minute, annealing at 54 °C for one minute, and elongation at 73 °C for one minute were performed. The products were analyzed on 5 percent denaturing polyacrylamide gels, and the frequency of the various alleles was determined after the dried gels had been subjected to autoradiography.
Mutational Analysis of the CD40 Ligand Gene
The cDNA was obtained after reverse transcription of total RNA extracted from peripheral-blood mononuclear cells stimulated with phorbol ester and ionomycin19. The CD40 ligand transcripts were amplified by a nested PCR and sequenced directly as described elsewhere10. For the determination of the fetal genotype, oligonucleotide primers surrounding the affected genomic region were synthesized with sequence data derived from a genomic clone of the CD40 ligand (unpublished data). Amplified DNA was then sequenced directly as described elsewhere10.
Results
CD40 Ligand Microsatellite Repeat
Molecular cloning of the human CD40 ligand revealed the presence of an extended dinucleotide repeat, (CA)32, in the 3' untranslated region of the cDNA8. Such simple repeated sequences of DNA, classified as microsatellite repeats, are widely dispersed in the mammalian genome,20 where they have attracted interest because of their high degree of polymorphism and, thus, their usefulness as genetic markers20. To determine whether the CD40 ligand CA repeat was polymorphic, oligonucleotide primers on either side of the repeat were used to amplify genomic DNA from 36 unrelated, healthy women by PCR. The CD40 ligand repeat proved to be polymorphic and informative (Table 1). Eight alleles were detected, for a 0.79 frequency of heterozygosity (i.e., 80 percent of the women had two different alleles). Thus, the CD40 ligand microsatellite repeat defined a highly polymorphic, intragenic marker suitable for linkage analysis in X-linked hyper-IgM immunodeficiency.
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Genomic DNA samples were prepared from affected and unaffected family members of four families with X-linked hyper-IgM immunodeficiency, and the pattern of segregation of the different alleles of the CD40 ligand microsatellite repeat was determined. Figure 1 shows the results in three families (a representative autoradiograph is shown for Family 3). For each family, the segregation of a particular allele in affected family members could be demonstrated. The validity of the CA typing was confirmed by sequence analysis of CD40 ligand transcripts in Family 3. In this family, two maternally related male cousins (Subjects III-1 and III-3) had the hyper-IgM syndrome. Microsatellite typing revealed the 164-base-pair (bp) mutant allele 2 in both subjects (Figure 1C). Direct sequence analysis of CD40 ligand transcripts showed a 10-bp deletion in the extracellular domain in both subjects (Figure 2), which results in an abnormal CD40 ligand devoid of CD40-binding activity10. Thus, the CD40 ligand CA repeat correctly identified affected subjects in families with the hyper-IgM syndrome.
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We have previously described a patient with the hyper-IgM syndrome who had a nucleotide point mutation (C to A) in the coding sequence of the CD40 ligand (Subject II-1 in Family 4). This resulted in a missense mutation (GCG to GAG) in which glutamic acid was substituted for alanine in the expressed protein at amino acid position 123, with the resultant loss of CD40-binding activity10. This mutation was identified in the mother of this patient as a heterozygous trait, thus confirming her as an obligate carrier with a 50 percent risk of transmitting the mutation to subsequent male offspring. During her second pregnancy, the mother wished to determine whether the fetus was affected with hyper-IgM immunodeficiency. A chorionic-villus sample was obtained at 12 weeks of gestation, and karyotypic analysis showed the fetus to be male. Typing of the CD40 ligand microsatellite was subsequently performed.
Analysis of the CD40 ligand repeat in the mother proved to be informative: two different-sized alleles (160 and 152 bp) were identified, allowing segregation analysis to be performed in her children (Figure 3). The 152-bp allele (allele 7) was present in the affected son (Subject II-1), identifying the mutant allele. Trophoblast DNA from the male fetus (Subject II-2) also displayed the 152-bp allele, identifying the fetus as affected with hyper-IgM immunodeficiency.
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We have identified an intragenic microsatellite repeat in the CD40 ligand gene, a highly polymorphic marker that is informative in 80 percent of women. Particular alleles of this gene are segregated in affected subjects from families with the hyper-IgM syndrome. We used this marker to make a prenatal diagnosis of X-linked hyper-IgM immunodeficiency in a male fetus at 12 weeks of gestation. This CD40 ligand microsatellite repeat fulfills the criteria required for a genetic screening test for this disease. Because this repeat structure resides in the 3' untranslated region of the CD40 ligand gene, it can be used to identify mutations regardless of their location in the CD40 ligand coding sequences. Moreover, we have recently determined that this dinucleotide repeat lies within 15 kb of the initiation codon of the CD40 ligand gene (unpublished data). Thus, the possibility of genetic recombination between the microsatellite repeat and the actual site of mutation is extremely small (on the order of 0.01 percent).
Patients with the hyper-IgM syndrome have considerable morbidity and an increased risk of premature death1. The intravenous administration of immune globulin has remained the mainstay of therapy, although it is not curative. One therapeutic alternative is bone marrow transplantation, but this option is often hampered by the lack of histocompatible donors. The identification of the CD40 ligand as the gene defective in X-linked hyper-IgM immunodeficiency may permit novel treatments involving replacement therapy with recombinant, soluble forms of the CD40 ligand. There will still be a need for genetic screening to allow prenatal diagnosis and to identify carriers of this disease. The CA repeat described here may be helpful in genetic counseling for X-linked hyper-IgM immunodeficiency.
Supported by grants from the Association Francaise contre les Myopathies, the Ministere de la Recherche et de la Technologie, the Caisse Nationale d'Assurance Maladie des Travailleurs Salaries, and INSERM.
Source Information
From INSERM Unite 132, Hopital Necker-Enfants Malades, Paris (J.P.D., S.M., A.F., G.S.B.), and the Glaxo Institute for Molecular Biology, Plan-les-Ouates, Geneva (J.-F.G., J.-Y.B.).
Address reprint requests to Dr. DiSanto at INSERM Unite 132, Hopital Necker-Enfants Malades, 149, rue de Sevres, 75743 Paris CEDEX 15, France.
References
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