A study of 82 extended HLA haplotypes in HFE-C282Y homozygous hemochromatosis subjects: relationship to the genetic control of CD8+ T-lymphocyte numbers and severity of iron overload

Abstract

Background: It has been recently demonstrated that CD8+ T-lymphocyte numbers are genetically transmitted in association with the MHC class I region. The present study was designed with the objective of narrowing the region associated with the setting of CD8+ T-lymphocyte numbers in a population of C282Y homozygous hemochromatosis subjects, in whom a high prevalence of abnormally low CD8+ T-lymphocyte counts has been described.

Methods: The study includes 43 C282Y homozygous subjects fully characterized both phenotypically and genotypically. Clinical characterization includes measurements of iron parameters at diagnosis (transferrin saturation and serum ferritin), total body iron stores and T-cell immunophenotyping determined by flow cytometry. Genetic characterization includes HLA class I alleles (A, B and C) and four additional microsatellite markers (D6S265, D6S2222, D6S105 and D6S2239) spanning 5 Megabases in the 6p21.3 region.

Results: Eighty-two extended C282Y carrying haplotypes were defined. Single-locus analysis revealed that the HLA-A region was associated with CD8+ T-cell numbers. Multivariate analysis showed that the combinations of the most common HLA-A alleles (HLA-A*03, -A*02 and -A*01) were associated with significantly lower numbers of CD8+ T-lymphocytes (0.30 +/- 0.14 x 106/ml), in comparison with subjects carrying only one copy of those alleles (0.46 +/- 0.19 x 106/ml) and subjects without any copy of those alleles (0.79 +/- 0.15 x 106/ml;p = 0.0001). No differences were observed in CD8+ T-cell counts among control subjects carrying the same combinations of HLA-A alleles (0.47 +/- 0.14; 0.45 +/- 0.21 and 0.41 +/- 0.17 x 106/ml, respectively), therefore not supporting a direct effect of HLA specificity but rather an indirect association with a locus close to HLA-A. Multivariate analysis showed that the combination of the most common HLA-A alleles also have an impact on the clinical expression of HH in terms of iron stores, in males(p = 0.0009).

Conclusion: The present study provides evidence supporting an inextricable link between extended HLA haplotypes, CD8+ T-lymphocyte numbers and severity of iron overload in hereditary hemochromatosis(HH). It gives additional information to better define a candidate region involved in the regulation of CD8+ T-lymphocyte numbers. A new evolutionary hypothesis concerning the inheritance of the phenotype of low CD8+ T-lymphocyte numbers associated with particular ancestral HLA haplotypes carrying the C282Y mutation and its implication on the clinical heterogeneity of HH is discussed.

Figure 1

Physical map of the polymorphic markers used in the present study and their relative localization (at scale) in relation to HLA-A, B and C and HFE.

Figure 2

Multi-locus map of C282Y carrying haplotypes from a region spanning 5 Megabases from HLA-B to D6S2239. The allele specificity in each locus is represented by numbers corresponding to the size of the amplified DNA fragments. Color boxes represent conserved haplotype areas. n.d.: not determined.

Figure 3

Mean values ± standard error of the mean of the CD8+ T-lymphocyte numbers according to the presence of different HLA-A alleles among haplotypes carrying the C282Y mutation. Haplotypes carrying the HLA-A*01 are 13, the A*02 are 16 and the A*03 are 39. The group of "All other A" specificities include: HLA-A*11, A*23, A*24, A*25, A*26, A*29, A*31, A*32, A*33 and A*68 and are 18.

Figure 4

Distribution of the CD8+ T-lymphocyte numbers (A) and of the total body iron store (TBIS) after correction for the effect of age (residuals of TBIS on age) (B), in males, according to the HLA-A genotype of the C282Y homozygous probands, which divide subjects in the three groups: 1.subjects with two copies of the most common HLA-A alleles (A*03, A*02 and A*01; n = 27); 2.subjects with only one copy of the most common HLA-A alleles (n = 14) and 3.subjects without any copy of those alleles (n = 2).