HLA and IgA deficiency in blood donors

We have identified 67 IgA deficient healthy blood donors in our region by systematic screening of 24,782 blood samples. HLA typing results on 36 of these donors indicated a significant association with both HLA-A1 and HLA-B14 antigens. The frequency of A29 and B8 antigens was also increased. However, B8 association may be secondarily involved due to linkage disequilibrium (e.g., A1-B8-DR3). The frequency of HLA-A1 and HLA-B8 antigens was increased in the group of IgA deficient donors who developed anti-IgA antibodies (53.9%) compared to those who did not (26.1%). Although the sample size appears to be too small to show a statistical significance (chi 2 = 2.76, 0.05 less than p less than 0.1), this is the first report to imply a possible HLA association with anti-IgA antibody formation by IgA deficient healthy individuals.     

Recurrent extended HLA haplotypes in children with selective IgA deficiency

HLA supratypes as well as serum IgG, IgA and IgM levels were determined in 44 children and adolescents with severe IgA deficiency (serum IgA less than 5 mg/dl) and in first degree relatives. Frequencies of the HLA alleles B14, DR1, DQW1, C4A2 and C4B2 were significantly higher in the IgA-deficient patients than in the controls. The most recurrent haplotype among patients was B14, DR1 (p less than 10(-4) preferentially associated with A33, A28 or A blank. The supratype B14, Bfs, C4A2, C4B2, DR1, DQW1 was present in a 14-fold higher frequency than in the controls, and strongly suggests the presence of a gene in the HLA region involved in the deficiency of IgA. The fact that this supratype was not always associated with IgA deficiency in the parents and that not all IgA-deficient subjects had this supratype is discussed. Severe IgA deficiency was found in four mothers (two of the four mother-child pairs shared the B14, DR1 haplotype); three other relatives (one father and two brothers) had partial IgA deficiency and did not have the B14, DR1, DQW1 haplotype.     

Association of chronic active hepatitis and HLA B35 in patients with hepatitis B virus

Fifty-one patients with chronic active hepatitis were typed for their HLA-A, B, C, and DR antigens. We observed a significant increase in the antigen frequency of HLA B35 in patients compared with controls.     

Functional assessment of a B cell defect in patients with selective IgA deficiency

The cellular basis of selective IgA deficiency was investigated by examining the terminal differentiation of B lymphocytes co-cultured with varying ratios of T lymphocytes in the presence of pokeweed mitogen. Eight patients were studied who had serum IgA concentrations <0·05 mg/ml, salivary IgA <0·01 mg/ml, and between 0·8 and 4% lymphocytes with surface IgA markers. Peripheral blood lymphocytes from patients and normal donors were separated into B cell (non T cell) and T cell fractions by E-rosetting. Microcultures were established at eleven B cell to T cell ratios from 100% B cells to 100% T cells. After 7 days, immunoglobulin in the supernatant fluid was measured by radioimmunoassay. Cultures containing patients' B cells and either autologous or allogeneic T cells produced very low or undetectable amounts of IgA. However, cultures from six out of eight patients contained cells with intracytoplasmic IgA. Secretion of IgM by the patients' B cells was identical to that of normal donors. Surprisingly, IgG production by patients' B cells was less than that produced by normal B cells especially in the mid-range ratios of the microcultures. Production of IgA, IgG and IgM by normal B cells from peripheral blood or tonsils was very similar in the presence of normal T cells or patients' T cells. In cultures containing optimal ratios of normal B cells, the patients' T cells not only did not suppress IgA production but also gave normal help for IgA production. It was concluded, on the basis of these studies, that a defect in patients with selective IgA deficiency was the functional inability of their B cells to produce normal amounts of IgA in vitro even when provided with normal allogeneic T cell help.     

HLA antigens in selective IgA deficiency: Distribution in healthy donors and patients with recurrent respiratory tract infections

HLA-A, B, C and DR typing was performed in healthy IgA deficient donors and in IgA deficient patients with recurrent respiratory tract infections. In healthy donors, a strong association with HLA B8 and DR3 was observed. In the patient group however, no statistically significant association with these antigens could be found. In contrast, an increased frequency of HLA B40 was noted. The preference for HLA B8/DR3 in healthy donors could not be attributed to higher levels of serum immunoglobulins since no major differences in the concentrations of IgM, IgG or the IgG subclasses were seen between the two groups.     

Molecular genetics of C4B deficiency in IgA nephropathy

The fourth component of complement (C4) occurs in two functionally distinct isotypes, C4A and C4B. The two closely linked genes are located on chromosome 6p, between HLA-B and -DR. Several reports have established complete C4B deficiency as the major genetic risk factor for IgA nephropathy (RR = 6.5; P = 0.0004). It is not clear whether this association derives from immune dysfunction related to the absent isotype or from another disease susceptibility gene closely linked to C4B. To help distinguish between these mechanisms, we examined the molecular basis of complete C4B deficiency in five patients with IgA nephropathy and eight healthy individuals.

C4 and Bf protein typing were performed by immunofixation electrophoresis of plasma. Genomic DNA was digested with several restriction enzymes, chosen to produce informative restriction fragment length polymorphisms (RFLPs). After electrophoresis and Southern blotting, digests were hybridized to a series of cDNA probes specific to the 5′ and 3′ ends of the C4 genes, the C4d region, and the adjacent 21-hydroxylase genes. Availability of DNA from family members allowed assignment of RFLPs to specific haplotypes.

The 10 C4B-deficient IgA nephropathy-associated haplotypes displayed seven different protein phenotype/RFLP patterns. Three haplotypes consisted of the common C4B/21-hydroxylase deletion on the Bf^*S, C4A^*3, C4B^*Q0 complotype. Two haplotypes were characterized by the C4A^*3,2 duplication, with two C4 genes present but a C4A protein being produced by the gene at the usual C4B locus. All of the remaining haplotypes had unique Bf, C4, and 21-hydroxylase patterns.

C4B-deficient IgA nephropathy patients display a variety of molecular genetic bases for their protein deficiency. This observation speaks against linkage of C4B deficiency with a locus encoding disease susceptibility and supports a primary role for the complement abnormality in this disease.     

IgA deficiency and autoimmunity

IgA is the most abundant immunoglobulin in the human body, and performs a very specialized role which involves mucosal immunity, development of tolerance and protection against infection. IgA is the key immunoglobulin in the respiratory and gastrointestinal tracts, which provide the most intimate interface between the environment and self. Normal levels of IgA are based on early studies consisting of only small numbers of patients. The international consensus definition of IgA deficiency is a level of 0.07 g/l after the age of four years in the absence of IgG and IgM deficiencies. The epidemiology of IgA deficiency reveals interesting variances between geographical regions – the incidence in Caucasians being much higher than that in Asians. IgA deficiency has also been found to co-exist with autoimmune diseases, allergies and malignancies. The association with autoimmunity is particularly interesting because it suggests a common genetic linkage that could potentially also explain the diversity in geoepidemiology. Both MHC and non-MHC associations have been described and the 8.1 haplotype has been significantly associated with autoimmunity in IgA deficiency patients over controls. Non-MHC genetic associations include IFIH1 and CLEC16A. The mutations leading to IgA deficiency have not been defined, but in some cases of IgA deficiency it has been suggested that the pathogenesis involves a failure in switched memory B cells that can lead to this cohort experiencing an increased incidence of recurrent bacterial infections or autoimmune diseases. Attempts to investigate the role of cytokines that can induce IgA synthesis in cells of patients with IgA deficiency, such as IL21 or the combination of CD40L/anti-CD40, IL-4 and IL10, are underway.     

Serum levels of IgA1 and IgA2 in children and in patients with IgA deficiency

Serum levels of IgA1 and IgA2 were measured by solid phase radioimmunoassay in samples from 110 children between 3 months and 10 years of age. Both IgA1 and IgA2 were detectable in all samples, and both IgA1 and IgA2 increased with increasing age. The percent of total serum IgA that was IgA2 did not change with age and was the same in samples from children (15.05 +/- 10.2%) as in samples from adults (15.86 +/- 7.98%). The proportion of serum IgA that was IgA2 was much less variable within sibships than within the group at large (P less than 0.005). In the 16 patients with IgA deficiency, the proportion of serum IgA that was IgA1 or IgA2 was highly variable. IgA2 constituted more than 50% of the IgA in 5 patients and less than 7% of the IgA in an additional 5 patients. These findings suggest that regulation of serum concentrations of IgA1 and IgA2 is complex and influenced by genetic factors and probably other unidentified factors.     

Association of the CD14 gene -159C polymorphism with progression of IgA nephropathy

The risk factors associated with the progression of IgA nephropathy (IgAN), the most common form of glomerulonephritis, are unclear. It has been suggested that CD14 signalling in response to various microbes affects the natural history of chronic inflammatory conditions. It has been hypothesised that variants in the promoter region of the CD14 gene might alter the expression of CD14, and this in turn could influence the progressive nature of IgAN.

PCR-RFLP was used to determine the polymorphism at the -159 site (T to C). The distribution of the CD14/-159 polymorphism was no different in patients with IgAN (n=216) compared to 171 healthy controls. After follow up for 86 months, it was found that an excess of the C genotype occurred in patients with progressive disease (p=0.03) and the risk of disease progression increased as the number of C alleles increased (p for trend = 0.002). The hazard ratio for progression in the patients with the CC genotype was 3.2 (p=0.025) compared with the patients possessing the TT genotype. After LPS stimulation, sCD14 was released more abundantly from the PBMCs of the TT subjects than from that of the CC subjects (p=0.006), even though mCD14 expression level was no different. In addition, the TT subjects released less IL-6 than the CC subjects after stimulation (p=0.0003). These results suggest that the CD14/-159 polymorphism is an important marker for the progression of IgAN and may modulate the level of the inflammatory responses.

     

Monogenic mutations associated with IgA deficiency

Introduction: For twenty years, two paradigms have been considered as the main genetic contributors to immunoglobulin A deficiency, including cytogenetic defects involving large chromosomal aberrations and an association with the human major histocompatibility complex (MHC) locus. However, an overview of recent studies suggests a role for several monogenic disorders in the development of this disease.

Areas covered: This review examines the concept of monogenic disorders for patients with IgA deficiency in order to identify the underlying pathogenic mechanism(s).

Expert commentary: A clinical/immunologic workup followed by targeted gene mutation analysis has been proposed for an approach to IgA deficient patients.     

Association of Mucosal Leishmaniasis with HLA

In the search for genetic variability in individual susceptibility to mucocutaneous leishmaniasis, a disease caused mainly by Leishmania (Viannia) braziliencis, HLA typing was performed on 43 patients presenting mucosal lesions and 111 matched controls. Antigen specificities of the HLA-A, -B, -C, -DR, and -DQ loci were determined and their frequencies in patients and controls were compared. There was a significant decrease in the frequency of HLA-DR2 [1 out of 38 (2.6%) patients vs. 29 out of 102 (28.4%) controls, corrected p value 0.004, relative risk 0.07, preventive fraction of the total population 0.26] as well as a significant increase of HLA-DQw3 [29 out of 38 (76.3%) patients vs. 43 out of 99 (43.4%) controls, corrected p value 0.006, relative risk 4.2, etiologic fraction of the population 0.58]. These results support participation of HLA class II molecules in individual susceptibility to mucocutaneous leishmaniasis and in the pathogenesis of metastatic, mucosal disease.     

T and B lymphocyte subpopulations and activation/differentiation markers in patients with selective IgA deficiency

Selective deficiency of immunoglobulin A (IgAD) and common variable immunodeficiency (CVID) are genetically closely related diseases, both of unknown pathogenesis. A plethora of abnormalities in lymphocyte subpopulations and expression of activation markers were repeatedly documented in CVID patients, while almost no data are available about lymphocyte subpopulations in IgAD patients. We determined basic lymphocyte subpopulations and those subpopulations that were reported to be abnormal in CVID patients (CD25, human leucocyte antigen (HLA)-DR CD45RA, CD45RO, CD27, CD28 and CD29 on both CD4(+) and CD8(+) cells, CD57 and CD38 on CD8(+) cells, CD21, CD27, IgM, IgD on B lymphocytes) in 85 patients with IgAD, 47 patients with CVID and in 65 healthy controls. Statistical analysis was performed by the Mann-Whitney U-test; significant P-values were determined by means of Bonferoni's correction. Our results showed an increase in the relative number of CD8(+) cells and a decrease in the absolute number of CD4(+) cells compared to healthy people, but similar abnormalities in CVID patients were much more expressed. IgAD patients had significantly decreased expression of HLA-DR and increased expression of CD25 on CD4(+) lymphocytes, also CD29 expression was decreased on CD8(+) cells, while other activation/differentiation markers on T cells (including the expression of CD45RA and CD45RO antigens) were not changed. There were no statistically significant abnormalities in B lymphocyte developmental stages in IgAD patients compared to healthy controls. Our observation showed that the majority of T and B lymphocyte subpopulation abnormalities described previously in CVID are not present in IgAD patients.