X-linked Hyper IgM (HIGM1) in an African kindred: the first report from South Africa
© Pienaar et al; licensee BioMed Central Ltd. 2003
Received: 05 September 2003
Accepted: 28 November 2003
Published: 28 November 2003
The objective of this study was to describe the clinical and molecular features of the first South African family with X-linked hyper-IgM syndrome (HIGM1).
Diagnoses were based on immunoglobulin results and the absence of CD40 ligand (CD40L) expression on activated T-cells. Complete molecular characterisation involved CD40L cDNA sequencing, and genomic DNA analysis by polymerase chain reaction amplification, restriction enzyme digestion and sequencing. A PCR-based diagnostic assay was established for carrier detection and prenatal diagnosis in this family.
There were originally six children, three males and three females. The eldest boy died after being diagnosed with hypogammaglobulinaemia, before HIGM1 was considered. This disorder was diagnosed in the second eldest boy at the age of 5 years, after failing to detect CD40L expression on his activated T-cells. A deficiency of CD40L was also confirmed in the youngest male at the age of 5 years. Both younger brothers have since died of infections relating to HIGM1. Molecular investigation showed that exon 3 was deleted from the CD40L mRNA of the affected males. Genomic DNA analysis identified a 1.5 kilobase deletion, spanning exon 3 and including extended flanking intronic sequence. Carrier status in the mother was confirmed by RT-PCR of her CD40L mRNA. Genetic analysis of the three female children was deferred because they were below the legal consenting age of 18 years. A PCR-based assay for genomic DNA was established for easy identification of female carriers and affected males in the future.
This study confirmed the diagnosis of HIGM1 in the first South African family to be investigated and identified a novel mutation in the CD40L gene.
Immunodeficiency with hyper-IgM was first described in 1960  and mapped to Xq26, in 1992, using DNA from families showing X-linked inheritance of the condition . This locus was known to contain the CD40 ligand (CD154) gene. Shortly thereafter, several groups independently showed that mutations in this gene caused the disorder, which is now known as Hyper IgM Type I (HIGM1) [3, 4]. The autosomal recessive forms took longer to delineate with two defects being described in the past three years. Hyper IgM Type II (HIGM2) is caused by mutations in the activation-induced cytidine deaminase (AID) gene, while Type III (HIGM3) is due to defects in the gene encoding CD40 [5, 6].
The clinical manifestations of HIGM1 include recurrent upper and lower respiratory tract infection, interstitial pneumonia, chronic diarrhoea, oral ulcers, sclerosing cholangitis and hepatitis. Less commonly arthritis, meningoencephalitis and tumours occur [7, 8]. Patients with HIGM1 are uniquely susceptible to interstitial pneumonia caused by Pneumocyctis carinii. Cryptosporidium parvum is frequently isolated from patients with chronic diarrhoea and ascending cholangitis. The immunodeficiency classically presents with elevated or normal IgM levels and low IgA, IgG and IgE concentrations. Neutropaenia is frequently documented. Treatment consists of prophylactic co-trimoxazole and intranvenous immunoglobulin replacement, and advice to boil water to prevent cryptosporidium infection. Granulocyte-colony stimulating factor may improve the neutropaenia. Despite optimal medical treatment prognosis is unfavourable as less than 30% survive beyond the 3rd decade of life. Bone marrow transplantation may be curative and is being increasingly considered Patients with advanced liver disease occasionally benefit from combined liver and bone marrow transplantation [9, 10].
The CD40L gene comprises 5 exons, 4 introns, and spans 12 kilobases. It encodes a type II transmembrane glycoprotein of 261 amino acids, which has a molecular mass of 39 kDa and is a member of the TNF superfamily. Domains of the protein include a short cytoplasmic tail, a transmembrane section and an extracellular region that shares homology with TNF-α. CD40L is mainly expressed by activated CD4+ T-cells [4, 11, 12] and is functional as a homotrimer. Other cells expressing CD40L include B cells, macrophages, monocytes, dendritic cells and endothelial cells. The interaction of CD40L on activated CD4 cells with CD40 on B-cells induces B-cell proliferation, immunoglobulin isotype switching, germinal centre formation and somatic hypermutation. Dysfunctional CD40 ligation thus prevents isotype switching from IgM, and results in the typical immunodeficiency [12, 13]. Failure to isotype switch, as well as failure to activate Kuppfer cells and pulmonary macrophages increases the risk for Pneumocystis carinii and Cryptosporidial infection . Autosomal forms of hyper-IgM syndrome are due to defects of B-cells, rather than T-cells .
The European CD40L defect database contains mutations scattered throughout the length of the gene. However, mutations are more common in the extracellularly located TNF-homology domain . A variety of missense, nonsense, splice-site, deletion and insertion mutations have been reported [15, 16]. In the present report we describe the characterisation of a novel deletion mutation in the first South African kindred with HIGM1.
CD40L expression on activated T cells was measured by flow cytometry as previously described .
Activation of T-cells and CD40L cDNA analysis
Peripheral blood mononuclear cells (PBMCs) (10 × 106) separated from heparinized blood by Ficoll isopaque density centrifugation, were incubated in 10 ml RPMI-1640 cell culture medium, 12% AB serum, with phorbol myristate acetate (PMA) (10 000 ng/ml) and Ionomycin (1.5 mg/ml) for 6 hours, to allow for activation of the T-cells. Total RNA was isolated and the mRNA fraction converted into cDNA, from which CD40L cDNA was amplified using CD40L specific primers. The primer pair used was: 5'-CTCTGCCAGAAGATACCATTTCAAC-3' (F) and 5'-TATGAAGACTCCCAGCGTCAGC-3' (R). The amplified cDNA was sequenced using the ABI BigDye sequencing method.
Clinical and laboratory data
Age at investigation
Immunoglobulin levels (initial)
Treatment and Outcome
IgM ↑, IgG ↓, IgA normal
4-weekly IVIG administered. Developed bronchiectasis. Died at age 17 months during an episode of pneumonia and acute-on-chronic respiratory failure
IgM normal, IgG ↓, IgA ↓
4-weekly IVIG and cotrimoxazole prophylaxis administered. Developed cryptococcal-associated ascending cholangitis / chronic liver disease. Died at the age of 7 years of an unknown cause
IgM ↑, IgG ↓, IgA normal
1 infusion of IVIG administered. Died at age 10 months due to interstitial pneumonia and acute respiratory failure
Nucleic acid analysis
To facilitate carrier detection we set up a PCR assay using primers flanking the deleted region. This assay gives a PCR product of 1741 bp from normal alleles and a product of 241 bp from the mutant allele. This is clearly shown in Figure 3b where PCR products from the patient and his carrier mother have been compared.
This deletion mutation is novel and has been submitted to the CD40L database with the following annotation: Reference sequence NT_011719, variant sequence bases 3083058–3084557 deleted .
In sub-Saharan Africa the diagnosis of primary immunodeficiency diseases is constrained by limited diagnostic facilities; few African centres have reported on these conditions. This manuscript is the first South African report of a kindred with HIGM1 to be fully characterised at the clinical and molecular level. While the immunology service at Red Cross Children's Hospital has been diagnosing primary immunodeficiency diseases for more than 30 years  a flow cytometric assay  for detecting CD40 ligand expression on activated T-cells was only established in 1998, facilitating the diagnosis of HIGM1 in the index family. This development occurred after the eldest, affected boy had died.
Low IgG concentration with either a normal or increased IgM concentration was present at the initial assessment of all three patients and is a fairly consistent feature of HIGM1 . However, the normal IgA concentration recorded in two of the affected males is not a usual feature, and it should not thus preclude consideration of HIGM1 in the differential diagnosis of males with hypogammaglobulinaemia. Although the second patient initially had a normal IgM concentration, 3 years later the IgM concentration was 1.5 times the normal upper limit. The elevated IgM concentration in these patients is not a genetically determined feature but rather reflects a chronic, poorly controlled primary response to the many infections that afflict these patients . Medical treatment did not prevent the early demise of the three children. Despite intravenous immunoglobulin therapy less that 30% of patients with HIGM1 live beyond the third decade as they also have an increased tendency to autoimmune disease and cancer [8, 19]. Improved outcome of bone marrow transplantation makes this therapeutic modality increasingly attractive in treating children with HIGM1 [9, 20].
The deletion of exon 3 from the CD40L gene not only results in the loss of 19 amino acids from the primary sequence but also alters the reading frame of the mRNA transcript beyond the exon2 / exon 4 coupling (Figure 2a), resulting in a premature termination codon 12 amino acid residues downstream. The final mRNA transcript is therefore shorter and carries a premature termination codon within the first third of the prescribed coding sequence. The introduction of premature stop codons in mRNA transcripts is known to often hasten their degradation in a process referred to as nonsense-mediated mRNA decay  and appears to be operative for this mutation. The amplification of CD40L cDNA from the carrier mother of our patient, clearly shows a marked reduction in staining intensity for the shorter and mutated transcript (Figure 2b) and is suggestive of a large discrepancy in PBMC template concentrations of the mRNA transcripts from the two alleles she carries.
Scrutiny of the CD40L mutation database reveals that large deletions are rarely encountered in affected subjects, showing the absence of repeat unit "hotspots" sometimes recorded at other gene loci . We have analysed the breakpoint and flanking sequences from our patient and find that the deletion has occurred at a short 4 bp direct repeat (GGCC), which is similar to the short direct repeats reported in deletions of the retinoblastoma gene .
Carrier detection in this family has only been extended to the mother of our patient where PCR confirmed her obligate carrier status (Figure 3b). This family has been counselled to delay screening of the three female siblings until they are older and are contemplating having children of their own.
In conclusion, advances in the fields of genetics, and cellular and molecular biology during the last decade have helped to clarify many aspects of the pathogenesis of the hyper IgM syndromes, and allowed us to completely characterise the HIGM1 mutation in this family. Recent progress should lead to a greater understanding of the spectrum of immunological and genetic disorders in sub-Saharan Africa.
List of abbreviations
complementary deoxyribonucleic acid
X-linked hyper IgM syndrome or Hyper IgM Type I
Hyper IgM Type II
Hyper IgM Type III
messenger ribonucleic acid
polymerase chain reaction
reverse transcriptase polymerase chain reaction
tumor necrosis factor
tumor necrosis factor-α
peripheral blood mononuclear cells
phorbol myristate acetate
The financial support of the Institute of Child Health, the UCT research committee and the Medical Research Council is gratefully acknowledged.
- Isreal-Asselain R, Burtin P, Chebat J: Un trouble biologique nouveau: L'agammaglobulinemie avec beta2-microglobulinemia (un cas). Bull Soc Med Hosp Paris. 1960, 76: 519-523.Google Scholar
- Padayachee M, Feighery C, Finn A, McKeown C, Levinsky RJ, Kinnon C, Malcolm S: Mapping of the X-linked form of hyper-IgM syndrome (HIGM1) to Xq26 by close linkage to HPRT. Genomics. 1992, 14 (2): 551-3.View ArticlePubMedGoogle Scholar
- Allen RC, Armitage RJ, Conley ME, Rosenblatt H, Jenkins NA, Copeland NG, Bedell MA, Edelhoff S, Disteche CM, Simoneaux DK, Fanslow WC, Belmont J, Spriggs MK: CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science. 1993, 259: 990-3.View ArticlePubMedGoogle Scholar
- Aruffo A, Farrington M, Hollenbaugh D, Li X, Milatovich A, Nonoyama S, Bajorath J, Grosmaire LS, Stenkamp R, Neubauer M: The CD40 ligand gp39 is defective in activated T cells from patients with X-linked hyper IgM syndrome. Cell. 1993, 72: 291-View ArticlePubMedGoogle Scholar
- Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, Catalan N, Forveille M, Dufourcq-Labelouse R, Gennery A, Tezcan I, Ersoy F, Kayserili H, Ugazio AG, Brousse N, Muramatsu M, Notarangelo LD, Kinoshita K, Honjo T, Fischer A, Durandy A: Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of hyper-IgM syndrome (HIGM2). Cell. 2000, 102: 565-575. 10.1016/S0092-8674(00)00079-9.View ArticlePubMedGoogle Scholar
- Ferrari S, Giliani S, Insalaco A, Al-Ghonaium A, Soresina AR, Loubser M, Avanzini MA, Marconi M, Badolato R, Ugazio AG, Levy Y, Catalan N, Durandy A, Tbakhi A, Notarangelo LD, Plebani A: Mutations in CD40 gene causes an autosomal recessive form of immunodeficiency with hyper IgM. Proc Natl Acad Sci USA. 2001, 98: 12614-12619. 10.1073/pnas.221456898.View ArticlePubMedPubMed CentralGoogle Scholar
- Levy J, Espanol-Boren T, Thomas C, Fischer A, Tovo P, Bordigoni P, Resnick I, Fasth A, Baer M, Gomez L, Sanders EA, Tabone MD, Plantaz D, Etzioni A, Monafo V, Abinun M, Hammarstrom L, Abrabamsen T, Jones A, Finn A, Klemola T, DeVries E, Sanal O, Peitsch MC, Notarangelo LD: Clinical spectrum of X-linked hyper-IgM syndrome. J Pediatr. 1997, 131 (1 pt 1): 47-54.View ArticlePubMedGoogle Scholar
- Notarangelo LD, Hayward AR: X-linked immunodeficiency with hyper-IgM (XHIM). Clin Exp Immunol. 2000, 120: 399-405. 10.1046/j.1365-2249.2000.01142.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Khawaja K, Gennery AR, Flood TJ, Abinun M, Cant AJ: Bone marrow transplantation for CD40 ligand deficiency: a single centre experience. Arch Dis Child. 2001, 84: 508-511. 10.1136/adc.84.6.508.View ArticlePubMedPubMed CentralGoogle Scholar
- Hadžic N, Pagliuca A, Rela M, Portmann B, Jones A, Veys P, Heaton ND, Mufti GJ, Mieli-Vergani G: Correction of the hyper-IgM syndrome after liver and bone marrow transplantation. N Engl J Med. 2000, 321: 320-324. 10.1056/NEJM200002033420504.Google Scholar
- Hollenbaugh D, Wu LH, Ochs HD, Nonoyama S, Grosmaire LS, Ledbetter JA, Noelle RJ, Hill H, Aruffo A: The random inactivation of the X chromosome carrying the defective gene responsible for X-Linked Hyper IgM Syndrome (XHIM) in female carriers. J Clin Invest. 1994, 94: 616-622.View ArticlePubMedPubMed CentralGoogle Scholar
- Clark LB, Foy TM, Noelle RJ: CD40 and its ligand. Adv Immunol. 1996, 63: 43-78.View ArticlePubMedGoogle Scholar
- Durie FH, Foy TM, Masters SR, Laman JD, Noelle RJ: The role of CD40 in the regulation of humoral and cell-mediated immunity. Immunol Today. 1994, 15: 406-411. 10.1016/0167-5699(94)90269-0.View ArticlePubMedGoogle Scholar
- Conley ME, Larché M, Bonagura VR, Lawton AR, Buckley RH, Fu SM, Coustan-Smith E, Herrod HG, Campana D: Hyper IgM syndrome associated with defective CD40-mediated B cell activation. J Clin Invest. 1994, 94: 1404-1409.View ArticlePubMedPubMed CentralGoogle Scholar
- The European CD40L defect database. [http://www.uta.fi/imt/bioinfo/CD40Lbase/]
- Notarangelo LD, Peitsch MC: CD40Lbase: A database of CD40 L gene mutations causing X-linked hyper-IgM syndrome. Immunol Today. 1996, 17: 511-516. 10.1016/0167-5699(96)30059-5.View ArticlePubMedGoogle Scholar
- O'Gorman MRG, Zaas D, Paniagua M, Corrochano V, Scholl PR, Pachman LM: Development of a rapid whole blood flow cytometry procedure for the diagnosis of X-linked hyper-IgM syndrome patients and carriers. Clin Immunol Immunopathol. 1997, 85: 172-181. 10.1006/clin.1997.4422.View ArticlePubMedGoogle Scholar
- Eley B, Beatty D: Primary immunodeficiency diseases in Cape Town. Allergy & Clinical Immunology International. 2000, 12: 267-270.View ArticleGoogle Scholar
- Arkwright PD, Abinun M, Cant AJ: Autoimmunity in human primary immunodeficiency diseases. Blood. 2002, 99: 2694-2702. 10.1182/blood.V99.8.2694.View ArticlePubMedGoogle Scholar
- Antione C, Muller S, Cant A, Cavazzana-Calvo M, Veys P, Vossen J, Fasth A, Heilmann C, Wulffraat N, Seger R, Blanche S, Friedrich W, Abinun M, Davies G, Bredius R, Schulz A, Landais P, Fischer A, European Group for Blood and Marrow Transplantation; European Society for Immunodeficiency: Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies report of the European experience 1968 – 99. Lancet. 2003, 361: 553-560. 10.1016/S0140-6736(03)12513-5.View ArticleGoogle Scholar
- Buhler M, Wilkinson MF, Muhlemann O: Intranuclear degradation of nonsense codon-containing mRNA. EMBO Rep. 2002, 3: 646-51. 10.1093/embo-reports/kvf129.View ArticlePubMedPubMed CentralGoogle Scholar
- Rudiger NS, Gregersen N, Kielland-Brandt MC: One short well conserved region of Alu-sequences is involved in human gene rearrangements and has homology with prokaryotic chi. Nucleic Acids Res. 1995, 23: 256-60.View ArticlePubMedPubMed CentralGoogle Scholar
- Canning S, Dryja TP: Short direct repeats at the breakpoints of deletions of the retinoblastoma gene. Proc Natl Acad Sci USA. 1989, 86: 5044-48.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/3/12/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.