ALBERT

Description: Identifying the genes and gene products relevant to physiological systems and creating opportunities to elucidate their function are essential first steps in understanding the pathophysiology of disease. To dissect the genetic variation underlying hematopoietic, cardiovascular, lung, and sleep dysfunction, we established a Center for Mouse Models of Heart, Lung, Blood and Sleep (HLBS) Disorders at The Jackson Laboratory as part of the NHLBI Program for Genomic Applications (PGA). The major goal of the JAX PGA is to enable researchers to link both single-gene mutations and quantitative trait loci (QTL) to gene function and disease. To achieve this goal, we are generating new mutations in mice by chemical (ENU) mutagenesis, and characterizing the common inbred mouse strains to detect existing genetic variation. Here, we report an extensive body of hematologically relevant strain characterization data and the establishment of new animal models. All strain characterization data is deposited into the Mouse Phenome Database (MPD, http://www.jax.org/phenome), also accessible via the JAX PGA website (http://pga.jax.org). Data for up to 48 inbred strains are currently available and include complete blood counts and coagulation profiles (PT, aPTT, fibrinogen). These data allow investigators to identify the most appropriate strains for (a) physiological testing; (b) drug development; (c) progenitors in QTL crosses; (d) sensitized mutagenesis screens; and (e) direct hypothesis testing. For example, to maximize the potential for successful QTL identification, parental strains that differ substantially in the phenotype of interest, at least 2 standard deviations (SD), should be selected. We used our strain survey data to select parental strains for identification of QTL for baseline WBC count, an important risk factor for sickle cell disease severity. The strains C57BLKS/J and SM/J have WBC counts of 12.6 ± 1.6 and 3.3 ± 0.8 x 103/μL, respectively, a difference much greater the 2 SD, indicating a high statistical power. We identified a highly significant QTL (LOD = 7) on chromosome 1 in an initial genome wide scan of 279 F2 animals. Moreover, the availability of extensive phenotypic data across the inbred strains in conjunction with the availability of saturated sslp and SNP maps has allowed us to identify QTL in silico. As an example of the utility of the MPD in hypothesis testing, a modifier gene associated with decreased VWF levels is present in 5 of the 6 MPD strains showing the highest aPTT levels (see abstract by Johnsen et al). In total, 44 different phenotypic projects, each consisting of large datasets, can be freely accessed through the MPD. The JAX PGA mutagenesis effort in C57BL/6J mice has likewise yielded valuable resources. Nearly 100 new mutant strains are in various stages of development, including strains with phenotypes of interest to the hematology community (e. g., anemia, thrombocytopenia, leukopenia, leukocytosis). These animal models and all other JAX PGA resources (protocols, software, QTL locations) are freely available to the scientific community.

Description: 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. Genome17:2006); 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., Genetics169:2005) 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.

Description: Defects in red blood cell (RBC) membrane skeleton components cause hereditary spherocytosis (HS). Clinically, HS varies significantly even among individuals with identical gene defects, illustrating the profound effects of genetic background on disease severity. We exploited a new spontaneous mouse model, wan, which arose on the inbred C3H/HeJ strain, to identify quantitative trait loci (QTL) that modify the HS phenotype. Homozygous wan mice have severe HS due to a complete deficiency of erythroid band 3. A QTL analysis of RBC count, hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin content (MCHC) was performed in wan/wan mice from an F2 intercross between C3H/HeJ+/wan and CAST/Ei+/+ F1 hybrids. Hematologic and survival data from C3H, CAST/Ei F2 wan homozygotes support the hypothesis that genetic modifiers significantly influence the band-3 null HS phenotype. Significant QTL were identified for the MCV trait only, suggesting that RBC membrane characteristics are a target for modifier gene action. The most significant quantitative trait locus, Hsm1 (hereditary spherocytosis modifier 1), localizes to mouse Chromosome 12 and is dominant. The peak LOD score was obtained with a marker for Spnb1 encoding erythroid β-spectrin, an obvious candidate gene. (Blood. 2004;103: 3233-3240)

Description: A substantial genetic contribution underlies variation in baseline peripheral blood counts. We performed quantitative trait locus/loci analyses to identify chromosome regions harboring genes influencing red cell hemoglobin concentration using the cell hemoglobin concentration mean (CHCM), a directly measured parameter analogous to the mean cell hemoglobin concentration. Fourteen significant loci (gene symbols Chcmq1-Chcmq14) were detected. Seven of these influenced CHCM in a sex-specific fashion, and 2 showed significant interactive effects (epistasis). For quantitative trait locus/loci detected in multiple crosses, confidence intervals were narrowed using statistical and bioinformatic approaches. Two strong candidate genes emerged and were further analyzed: adult β-globin (Hbb) for Chcmq3 on Chr 7, and transferrin (Trf) for Chcmq2 on Chr 9. High and low allele parental strains in crosses detecting Chcmq3 segregate 100% with the known ancestral haplotype blocks, hemoglobin (Hb) diffuse (Hbbd) and Hb single (Hbbs), respectively. Hbbd consists of nonidentical major and minor polypeptides and exhibits an increased positive charge relative to Hbbs due to the net loss of 2 negative residues in the Hbbdminor polypeptide, resulting in a pI of 7.85 versus 7.13. Thus, as shown in human erythrocytes, positively charged Hbs are associated with cell dehydration and increased CHCM in mouse erythrocytes.