One cohort of healthy children and one of adults were recruited and measured for the Ig serum levels. Individuals were divided in three groups with low, medium and high serum levels with respect to the Ig median of each cohort (Additional file
1: Table S1 and in the Methods). The Ig levels were all in the normal range. Table
1A-B shows the allelic frequencies for both cohorts within each Ig class and reports that hs1.2 alleles are associated with Ig serum levels in children, but not in adults (Additional file
2: Figure S1 dot-plots the data summarized in both Table
1 and Additional file
1: Table S1). In fact, the comparison of Low (L) vs High (H) and Medium (M) vs H serum levels of Ig shows highly significant different allelic frequencies (constantly p<9.9E-08), only in the population of children. Similar variations of *1 and *2 allele frequencies in children were observed in each Ig class of young cohort: the *1 allele frequency decreases from the range of 41%-49% in L and M groups to the range of 2%-5% in H group and, on the contrary, *2 allele frequency increased from the range of 39%-49% in L and M groups to the range of 79%-82% in H group. It is remarkable that no young subjects homozygous for *1 allele were included in the H groups of IgM and IgG, and only one young subject was present in the H group of IgA. The frequencies of 2/2 homozygous genotype in children of L and M groups were between 1.7%-13.3%, while the H group of each Ig class was comprised between 60.0%-66.6%. In children and adults, the medians of Ig level were very similar, in agreement with the ranges reported in literature [
1] (see Additional file
1: Table S1). Nevertheless, the adult cohort did not show the same level and significance of allelic frequency variations than children when subdivided for Ig levels in L, M, and H groups (Table
1A; 0.69 < p < 0.92). Consequently the probability of one subject to be in one group of Ig level and also in the same group for the other Ig classes is very high in children, but not in adults (see Additional file
1: Table S2). The genetic association between Ig concentrations and hs1.2 polymorphism suggests likely age-related changes in Ig expression for children homozygous for *1 or *2 allele. Therefore, we followed up the Ig levels of those children for three years. As reported in Table
2, after three years (from five to eight) *1 allele homozygous children showed an increase of IgG and IgA levels, while *2 allele homozygous children showed a decrease in all Ig levels. We also investigated if B cell subpopulations of peripheral blood were similar in two homozygous groups by flow cytometric analysis, using IgM and IgD surface cell markers for the distribution of naive, marginal zone, IgM memory and switched memory B cells (Additional file
1: Table S3). Children homozygous for *1 and *2 alleles show no differences in the distribution of B cell subtypes, suggesting moreover that the number of B subtypes of circulating cells does not influence IgM concentration in individual with different genotype (Additional file
1: Table S3).
Table 1
Allelic frequencies of hs1.2 enhancer in children (A) and adults (B) divided for the serum levels of Ig
Children (f)
|
ALLELE 1
| 0.47±0.03 | 0.44±0.04 | 0.02 ± 0.01 | 0.48 ± 0.05 | 0.41 ± 0.04 | 0.05 ± 0.02 | 0.49 ± 0.04 | 0.40 ± 0.06 | 0.05 ± 0.03 |
ALLELE 2
| 0.43’ ± 0.04 | 0.46 ± 0.04 | 0.81 ± 0.04 | 0.44 ± 0.03 | 0.47 ± 0.06 | 0.79 ± 0.04 | 0.39 ± 0.05 | 0.49 ± 0.04 | 0.82 ± 0.03 |
ALLELE 3
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
ALLELE 4
| 0.1 ± 0.03 | 0.1 ± 0.02 | 0.17 ± 0.03 | 0.08 ± 0.02 | 0.12 ± 0.03 | 0.16 ± 0.03 | 0.12 ± 0.03 | 0.11 ± 0.03 | 0.13 ± 0.02 |
|
n.
| 60 | 70 | 60 | 60 | 70 | 60 | 60 | 70 | 60 |
B
| | | | | | | | | | |
Adults (f)
|
ALLELE 1
| 0.29 ± 0.03 | 0.32 ± 0.03 | 0.25 ± 0.02 | 0.30 ± 0.02 | 0.30 ± 0.02 | 0.26 ± 0.02 | 0.34 ± 0.03 | 0.26 ± 0.04 | 0.27 ± 002 |
ALLELE 2
| 0.56 ± 0.04 | 0.54 ± 0.02 | 0.58 ± 0.03 | 0.54 ± 0.04 | 0.55 ± 0.06 | 0.58 ± 0.04 | 0.52 ± 0.04 | 0.58 ± 0.03 | 0.57 ± 0.07 |
ALLELE 3
| 0 | 0.01±0.01 | 001±0.01 | 0.01±0.01 | 0.01±0.01 | 0.01±0.01 | 0.01±0.01 | 0 | 0.01±0.01 |
ALLELE 4
| 0.15±0.01 | 0.13 ± 0.02 | 0.16±0.01 | 0.15±0,01 | 0.14±0.01 | 0.16±0.02 | 0.13 0.13 0.03 | 0.16±0.01 | 0.15±0.02 |
|
n.
| 110 | 120 | 110 | 110 | 120 | 110 | 110 | 120 | 110 |
Table 2
Ig median follow-up from 5 to 8 years
1/1
| 10 | 98.5±0.07 | 94.5±0.05 | 4.94E-01 | 880.5±0.12 | 1029.5±0.14 | 1.31E-01 | 86.5±0.04 | 102±0.06 | 9.86E-02 |
2/2
| 30 | 217.5±0.11 | 1030 ± 0.09 | 1.09E-11 | 1674.5±0.16 | 979±0.13 | 1.98E-13 | 208±0.12 | 105.5±0.08 | 1.29E-13 |
Previous evidence showed the association of Ig levels with 3’RR1 enhancer hs1.2 polymorphism in autoimmune diseases. In these experiments we studied the role of hs1.2 polymorphism in physiological conditions in healthy subjects. Our results show that in children there is a significant correlation between different hs1.2 alleles and Ig expression levels, suggesting that hs1.2 polymorphism might primarily influence the concentration of Ig classes in serum during childhood. Moreover the follow-up of the children homozygous for the *1 allele showed that, after the fifth year of age, the Ig median values for all three classes increase; instead, in subjects homozygous for the *2 allele the values strongly decrease. This trend leads to the final adult equilibrium observed where no correlation is shown with Ig levels and hs1.2 alleles.