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Diagnosis of hyper-IgM syndromes, specifically X-linked hyper-IgM (HIGM1) and autosomal recessive hyper-IgM type 3 (HIGM3)
Evaluation of isotype class-switching defects
Hyperimmunoglobulin M (hyper-IgM) syndromes are a collection of primary humoral immunodeficiencies characterized by recurrent infections along with low serum IgG and IgA, and normal or elevated IgM. Over the course of the last several years, at least 5 genetic defects have been shown to be associated with this group of immunodeficiencies.(1-3) These genetic defects include mutations that affect:
-The costimulatory molecule, CD40LG, induced on activated T cells
-The CD40LG receptor, CD40, expressed constitutively on B cells
-Activation-induced cytidine deaminase (AID or AICDA), involved in somatic hypermutation (SHM) and isotype class-switching
-Uracil DNA glycosylase (UNG), also involved in isotype class-switching and partially in SHM
-NF-kappa B essential modulator (NEMO), also known as IKK gamma, which modulates NF-kappa B function(2,3)
The mutations that occur in the CD40LG and CD40 genes are associated with X-linked hyper-IgM (type 1) and autosomal recessive hyper-IgM (type 3), respectively. Patients with mutations in either of these 2 genes are particularly prone to infections with opportunistic pathogens, such as Pneumocystis jiroveci, Cryptosporidium parvum, and Toxoplasma gondii.(4)
All of the hyper-IgM syndromes (except those due to UNG defects and a hitherto undefined autosomal recessive [non-type 3] hyper-IgM) are associated with defects in isotype class-switching and SHM.(4) In the undefined autosomal recessive hyper-IgM there is no SHM defect, and in UNG deficiency there is biased SHM.(4) The impairment in isotype class-switching leads to the increased IgM levels with corresponding decrease in the "switched'' immunoglobulins such as IgG, IgA, and even IgE.
In the adult patient, hyper-IgM syndromes can overlap clinically with common variable immunodeficiency (CVID). However, patients with CD40LG (X-linked hyper-IgM; HIGM1) and CD40 (hyper-IgM type 3; HIGM3) mutations invariably present in infancy with upper and lower respiratory tract infections and opportunistic infections as previously described. HIGM1 is the most common of all the hyper-IgM syndromes described thus far, while HIGM3 is much rarer.
Intermittent neutropenia is common in HIGM1 and has also been reported for HIGM3. Both diseases show significant decreases in class-switched memory (CD27+IgM-IgD-) B cells, corresponding to profound reductions in serum IgG and IgA levels. Peripheral T-cell subsets are normal, though in HIGM1 the number of CD45RO+ memory T cells is reduced. T-cell lymphocyte proliferative responses to mitogens are normal in both HIGM1 and HIGM3, while responses to specific antigen are abnormal in HIGM1 and normal in HIGM3.
TBBS / T- and B-Cell Quantitation by Flow Cytometry and IABC / B-Cell Phenotyping Screen for Immunodeficiency and Immune Competence Assessment, Blood evaluate isotype class-switching defects with identification of various memory B-cell subsets, including class-switched memory B cells. The other components of this panel include the CD40LG XHIM / X-linked Hyper IgM Syndrome, Blood, and CD40 / B-Cell CD40 Expression by Flow Cytometry, Blood, which is the CD40 assay for HIGM3.
The absolute counts of lymphocyte subsets are known to be influenced by a variety of biological factors, including hormones, the environment, and temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells increase between 8:30 a.m. and noon with no change between noon and afternoon. Natural Killer (NK)-cell counts, on the other hand, are constant throughout the day.(5) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(6,7,8) In fact, cortisol and catecholamine concentrations control distribution and therefore, numbers of naive versus effector CD4 and CD8 T cells.(6) It is generally accepted that lower CD4-T cell counts are seen in the morning compared to the evening(9) and during summer compared to winter.(10) These data therefore indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.
The appropriate age-related reference values will be provided on the report.
An interpretive report will be provided.
The absence of CD40LG on activated T cells is consistent with X-linked hyper-IgM syndrome (HIGM1). The presence of CD40LG on activated T cells is not consistent with HIGM1. The presence of a positive and negative population for CD40LG is consistent with HIGM1 carrier status (mosaic).
Negative CD40-muIg staining is consistent with HIGM1. Positive CD40-muIg staining is not consistent with HIGM1. Some patients (approximately 20%) show absent (negative) staining with the CD40-muIg antibody, while there is positive staining for surface CD40LG on activated T cells. This dichotomy is due to the presence of specific mutations in the CD40LG gene that permit normal surface expression of the protein but abrogate function. Therefore, measurement of the receptor-ligand binding function using the chimeric CD40-muIg antibody improves the specificity of the assay, enabling identification of CD40LG-deficient patients who would be missed otherwise.
The absence of CD40LG protein expression or CD40-muIg binding is considered confirmatory for HIGM1. Genetic testing is not necessary to confirm the diagnosis, but may be performed to identify the specific mutation involved.
The absence of CD40 on B cells is consistent with autosomal recessive hyper-IgM type 3 (HIGM3). The presence of CD40 on B cells is not consistent with HIGM3.
Reduced or absent class-switched memory B cells (CD27+IgM-IgD-) is consistent with a defect in isotype class-switching. Normal numbers of class-switched memory B cells indicates the lack of an isotype class-switching defect.
The result for the CD40LG (X-linked hyper IgM) test is only valid when there is normal expression of CD69 upon T-cell activation.
Reduction of class-switched memory B cells is also seen in a significant proportion of patients with common variable immunodeficiency. Clinical correlation is essential to establishing the appropriate diagnosis.
Timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets. See data under Clinical Information.
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