Genetic Evidence That Sequence Variation at the β-Globin Locus Underlies Differences in Cell Hemoglobin Concentration and Cell Hydration in Single (Hbbs) Vs. Diffuse (Hbbd) Inbred Mouse Strains: Implications for Inherited Anemias

Abstract

Erythrocyte dehydration is a distinguishing feature of hematological diseases including sickle cell anemia and hereditary spherocytosis (HS). Identification of genes influencing hydration status will increase our understanding of red cell pathologies and provide new targets for risk assessment and drug development. We mapped quantitative trait loci (QTLs) influencing red cell hydration using the cell hemoglobin concentration mean (CHCM) as a marker of hydration status. CHCM (analogous to the calculated mean cell hemoglobin content, MCHC) was obtained using an Advia 120 whole blood analyzer, which provides a direct measure of hemoglobin concentration on a cell-by-cell basis. We established seven F2 intercrosses and one backcross and phenotyped 200–500 offspring from each cross at 8–10 weeks of age. Genome-wide scans were performed to detect single loci associated with CHCM (main effect QTL) using the R/qtl software (www.rqtl.org). Significance thresholds were determined by permutation testing (1000 permutations). A highly significant locus was identified on chromosome 7 in 6 of the 8 crosses (maximum LOD score of 82) with a 95% confidence interval (CI) spanning from approximately 90–129 mega base pairs (Mb). Previously we identified two QTL for CHCM (Peters et al., Mamm. Genome 17: 2006); hence, we have designated the chromosome 7 locus Chcmq3 (CHCM quantitative locus 3). Chcmq3 has a significant impact on the baseline CHCM; it alone accounts for 48% of the variance of the trait. The 95% CI was subsequently narrowed using statistical and bioinformatic tools. First we performed combined cross analysis (Li et al., Genetics 169: 2005) where genotype data from multiple crosses in which the same QTL is detected is re-coded to a phenotype-based designation based on allele effects and re-analyzed as a single dataset to increase statistical power. This analysis reduced the CI to 7 Mb containing 289 genes. Using a dense single nucleotide polymorphism (SNP) map, including imputed SNPs (Center for Genome Dynamics, http://cgd.jax.org) and phenotypic data from 32 inbred strains, we performed interval-specific haplotype analysis and haplotype association mapping to eliminate regions identical by descent and obtain a final interval of 0.38 Mb containing 30 candidate genes. Twenty one were olfactory receptor (Olfr) genes, poor biological candidates, leaving 9 candidates including the beta-globin cluster. Two major ancestral haplotype blocks span the beta globin cluster, and the parental strains contributing the high allele in each cross (129S1/SvImJ, DBA/2J, NZW/LacJ, WSB/EiJ, MRL/MpJ, CBA/J) are of the hemoglobin-diffuse electrophoretic variant (Hbbd), while the low allele strains (C57BL/6J, SM/J) are hemoglobin-single (Hbbs) variants. Notably, examination of the Mouse Phenome Database (www.jax.org/phenome, project Peters1) reveals that Hbbd strains all have relatively high CHCM values, while Hbbs strains have lower CHCM values. Moreover, in the two crosses in which Chcmq3 was not detected, the parental strains shared the same haplotype at the beta globin locus. As a QTL can not be detected in the absence of genetic variation in the underlying gene, this observation supports beta globin as a strong candidate gene for Chcmq3. Furthermore, in preliminary quantitative PCR studies, strain 129S1/SvImJ, a diffuse variant, showed higher expression of adult beta globin, Hbb-b1, in the spleen than C57BL/6J (single variant). The data suggest that polymorphisms in beta globin itself modify hemoglobin concentration in mice and reinforce the notion that hemoglobin charge and/or structure may directly or indirectly modulate membrane ion transport and erythrocyte hydration in both mice and humans.