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J Clin Immunol DOI 10.1007/s10875-008-9219-y Cellular and Molecular Characterisation of the Hyper Immunoglobulin M Syndrome Associated with Congenital Rubella Infection Rohan Ameratunga & See-Tarn Woon & Wikke Koopmans & John French Received: 29 April 2008 /Accepted: 9 June 2008 # The Author(s) 2008 Abstract Introduction Introduction The hyper-immunoglobulin M syndrome (HIM) is a rare group of immune deficiency disorders The hyper-immunoglobulin M syndrome (HIM) is a group characterised by normal or increased serum IgM with of immune deficiency disorders characterised by normal or normal or reduced IgG, IgA and IgE. elevated serum levels of IgM and normal or reduced IgG, Materials and Methods We have undertaken detailed IgA and IgE [1]. Patients with the X-linked form of the cellular and molecular studies in a 53-year-old man with disorder (XHIM) are susceptible to Pneumocystis jiroveci HIM as a result of congenital rubella. and Cryptosporidium parvum [2]. XHIM occurs when Results No mutations were detected in the CD40 ligand, mutations in the CD40 ligand (CD40L) prevents B cells activation-induced cytidine deaminase and uracil DNA from undergoing immunoglobulin class switching [3]. glycosylase. His T-cell responses to lectins and antigens However, some HIM patients have normal CD40L se- were normal. Flow cytometry confirmed the presence of quence and function [4, 5]. Mutations in activation-induced CD40 ligand on activated T cells. Most CD40-dependent cytidine deaminase (AICDA), uracil DNA glycosylase functions that were tested, including B-cell proliferation, (UNG) and CD40 can also lead to HIM as a result of isotype switching and production of memory B cells, were impaired immunoglobulin class switching [6–9]. normal. CD40/IL4 dependent rescue from anti-IgM- A small proportion of patients with congenital rubella induced apoptosis was impaired. develop a syndrome with recurrent infections and an Conclusion The detection of cell-surface IgG but lack of immunoglobulin profile similar to that of XHIM [10–13]. serum IgG indicated that he may have an antibody secretion With the advent of widespread immunisation, congenital defect. rubella has become a rare disorder in developed countries. Most of these patients were described before the wide- Keywords Hyper-IgM . rubella . non X-linked . spread availability of sophisticated molecular diagnostic isotype switching tools. In this report, we describe a patient with congenital rubella who developed HIM. He has normal CD40L sequence and function. No mutations were found in the AICDA and UNG genes. Several CD40 dependent B-cell * : : R. Ameratunga ( ) S.-T. Woon W. Koopmans functions were normal. He can generate switched memory Department of Virology & Immunology, Auckland City Hospital, Auckland, New Zealand B cells. Immunoglobulin isotype switching was observed e-mail: [email protected] both in vivo and in vitro. CD40/IL4 dependent rescue from anti-IgM-induced apoptosis was impaired. We postulate J. French that the patient may have a defect in immunoglobulin Department of Cardiology, Liverpool Hospital and University of New South Wales, secretion since cell surface IgG was detected despite low Sydney, Australia serum IgG. J Clin Immunol Materials and Methods number of infections. Family history revealed no relatives with recurrent infections or unexplained deaths in early Patient This study was approved by the University of childhood. The patient’s sister and her son and daughter are Auckland ethics committee. Patient M1 is a 53-year-old in good health. man born at term to non-consanguineous parents. His Apart from chronic tiredness and chronic sinusitis, he mother suffered a rash during pregnancy and was diagnosed currently maintains reasonable health. Although he has a as having rubella. The patient was born with impaired chronic cough, CT scans of the thorax failed to show any vision and sensorineural hearing loss. He requires a hearing evidence of bronchiectasis. Referral to the immunology aid. At the age of 3 years, he presented with recurrent upper clinic at the Auckland Starship Hospital in 1992 established and lower respiratory tract infections and was found to be the presence of grossly elevated polyclonal IgM consistent severely hypogammaglobulinemic. In 1961, the serum with HIM. IgM levels of up to 15 g/L (normal range 0.4– gamma globulin fraction was absent. In 1984, the IgG 2.5 g/L) were noted on several occasions. A graphical was below 2.5 g/L (normal range 8–16 g/L). He was depiction of his immunoglobulin levels are shown in Fig. 1. commenced on intramuscular and subsequently intravenous High doses of IVIG (1 g/kg per month) were given to the immunoglobulin (IVIG), which led to a decrease in the patient and another patient with XHIM, in order to reduce Fig. 1 Immunoglobulin levels XHIM for patients XHIM and M1 from 1987 to 1993. The increasing IgG levels reflect 20 increased monthly IVIG doses. The top panel shows the immu- 18 noglobulin profile for the patient 16 with XHIM. The bottom panel shows the immunoglobulin pro- 14 file for patient M1. Note that the data from infusion 21 may have 12 IgM been a laboratory error. The gap 10 IgA at infusion 37 represents a g/L period where data was not 8 IgG available. High-dose IVIG was 6 commenced at this time. Normal ranges IgG (8–16 g/L), IgM 4 (04–2.5 g/L), IgA (0.8–4.0 g/L) 2 0 1 4 7 1013161922252831343740434649525558 infusion Patient M1 16 14 12 10 IgM 8 IgA g/L IgG 6 4 2 0 1 3 5 7 9 111315171921232527293133353739414345474951 infusion J Clin Immunol B-cell proliferation and thus minimise the risk of B-cell pokeweed mitogen (PWM), phytohaemagglutinin (PHA), malignancy. A progressive reduction in IgM was noted in concanavalin A (ConA) and calcium ionophore A23187 the patient with XHIM. In contrast, patient M1 had further (Sigma, St. Louis, MO), anti-IgG4 (Unipath, Birmingham, increases in his IgM level. Serum and salivary IgA and UK), anti-IgM, IgG4 and biotin-conjugated anti-IgG4 anti- serum IgE were undetectable. Serum and urine electropho- body (Sigma Immunochemicals). Anti-CD3 monoclonal resis on multiple occasions excluded a monoclonal gamm- antibody (OKT3) was purchased from the Auckland Hospital opathy. His absolute and percentage CD4 and CD8 T-cell Pharmacy. CD40L trimer and cytokines were gifts from numbers were within normal range. Immunex Research and Development Corporation. All other antibodies were from Beckman Coulter (Fullerton, CA). Reagents Reagents were purchased from the following sources: tetanus toxoid (Lederle-Praxis Biologicals Inc, Pearl Isolation of peripheral blood mononuclear cells Peripheral River, NY), diphtheria toxoid (Statens Seruminstitut, blood mononuclear cells (PBMC) were isolated from a Denmark), Candida antigen (Greer Labs, Lenoire, NC), Ficoll–Hypaque gradient as previously described [14]. Fig. 2 Untreated (panel I) and IIl PMA/calcium ionophore a 4 A23187 treated (panel II) T cells 4 10 from patient M1 (a), patient 10 with XHIM (b) and healthy 3 3 10 control (c) were stained for cell 10 surface CD40L expression 2 2 10 10 1 1 10 10 CD40L PE unstimulated 0 0 10 10 100 101 102 103 104 100 101 102 103 104 CD3 FITC unstimulated CD3 FITC stimulated b 4 4 10 10 3 3 10 10 2 2 10 10 1 1 10 10 0 CD40L PE unstimulated 0 10 10 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 CD3 FITC unstimulated CD3 FITC stimulated c 4 4 10 10 3 3 10 10 2 2 10 10 1 1 10 10 CD40L PE unstimulated CD40L PE stimulated CD40L PE stimulated CD40L PE stimulated 0 10 0 10 100 101 102 103 104 100 101 102 103 104 CD3 FITC unstimulated CD3 FITC stimulated J Clin Immunol Genetic Studies Peripheral blood mononuclear cells were incubated for 1 h at 37°C prior to the addition of 1:1,000 stimulated for 16 h with phorbol ester (PMA) (500 ng/ml) biotin-conjugated anti-IgG4 antibody. Following a 1-h and PWM (10 μg/ml) prior to extraction of RNA. Comple- incubation at 37°C, horseradish peroxidase-conjugated strep- mentary DNA was synthesised following RNA extraction. tavidin was added before development with o-phenylene The CD40L gene was amplified in two overlapping frag- diamine. Serum and salivary immunoglobulin levels were ments. The polymerase chain reaction (PCR) products were detected by nephelometry. then ligated into the TA vector system (Promega, Madison, WI) and competent Escherichia coli cells were transformed. Flow Cytometry Peripheral blood mononuclear cells were Colonies containing inserts were selected and subjected to stimulated for 16 h with PMA (500 ng/ml) and calcium bidirectional sequencing of M13 cloned products. UNG and ionophore A23187 (50 ng/ml) prior to assessing CD40L AICDA were amplified as follows: 3 min at 95°C; 40 cycles expression on a Becton-Dickinson FACSCAN flow of 30 s at 95°C, 30 s at 57°C, 50 s at 68°C; 72°C, 5 min. We cytometer. Naïve, memory and switched memory B aligned the three gene sequences with published wild-type cells were phenotyped with anti-CD19-APC, anti- sequences [8, 9, 14] using SeqManII 5.01 (DNASTAR, CD27-FITC and anti-IgD-PE. Naïve and memory CD4 Madison, WI). and CD8 T cells were quantified with CD45RA-FITC, CD45RO-APC and CD62L-PE antibodies. A healthy T-cell Proliferation Assays T-cell proliferation was deter- control and a patient with well-characterised XHIM [14] mined by responses to T-cell-dependent lectins (PHA, were used for comparison. This patient was previously ConA, PWM) and OKT3 as well as a panel of soluble shown to have a complex splicing defect of the CD40 antigens (Candida, tetanus and diphtheria) as previously ligand. described [15].