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. 2023 Mar 1;108(3):905-908.
doi: 10.3324/haematol.2022.281767.

Identification of multiple genetic loci associated with red blood cell alloimmunization in mice

Arijita Jash  1 , Heather L Howie  2 , Ariel M Hay  1 , Chance John Luckey  2 , Krystalyn E Hudson  3 , Peter C Thomson  4 , Sarah J Ratcliffe  5 , Mark Smolkin  5 , James C Zimring  6
Affiliations

Identification of multiple genetic loci associated with red blood cell alloimmunization in mice

Arijita Jash et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Genetically distinct mouse strains have different tendencies towards red blood cell alloimmunization. Each of the indicated strains were transfused with HOD (A) or KEL-K2Lo (B) red blood cells (RBC) and IgG responses were tested in serum at 21 days after transfusion. Serum was incubated with alloantigen expressing RBC followed by a fluorescently labeled secondary antibody and median fluorescence intensity (MFI) was determined by flow cytometry. As both alloantigen transgenic mice are on a B6 background, serum was also incubated with B6 RBC and the MFI subtracted from values of alloantigen expressing target cells. Major histocompatibility complex (MHC) haplotypes of the mice used are [A/J (H-2a), AKR/J (H-2k), Balb/cByJ(H-2d), BTBR(H-2b), C57BL6/J(H-2b), C3H/HeJ(H-2k), DBA/2J(H-2d), FVB/NJ(H-2q), KK/HiJ(H-2b), NOD/ShiLtJ(H-2g7), 129S1/SVImJ(H-2b), 129SX1/SvJ(H-2b) ]. No antibodies to background antigens on B6 RBC were detected and the MFI of B6 RBC was subtracted from the MFI of target RBC expressing the indicated alloantigen. Negative values for IgG were determined to be non-biologically relevant and were thus set to a value of zero. The combined results of 3 different experiments are shown, with samples sizes ranging from 13-15 mice. Statistical significance is defined as a Sidak-Bonferroni adjusted P<0.05 in pairwise comparisons estimated from a two-part model (logit & log-linear links) adjusted for experiment – each strain is compared to B6.H2d. Statistical significance is indicated by (*P=<0.05) and (**P<0.001). No experimental results that were obtained with this approach that were excluded from this figure. HOD and KEL-K2Lo mice were bred in the University of Virginia vivarium, all recipient mice were purchased from The Jaxson Laboratory (Bar Harbor, ME) and all procedures were carried out in compliance with approved IACUC protocols.
Figure 2.
Figure 2.
Identification of multiple genetic loci that associate with red blood cell alloimmunization. 156 F2 mice were each transfused with KEL-K2Lo red blood cell (RBC) and alloantibodies were measured at 21 days post transfusion (A). For each animal, expression of NK1.1 was determined on splenocytes by flow cytometry (data not shown). Quantitative trait locus (QTL) analysis was carried out using alloimmunization to RBC (B) or NK1.1 expression (C) as the trait. Starting with a total of 11,125 single nucleotide polymorphisms (SNP), 3,220 differed between the B6 and 129 strains, with 3,118 remaining after filtering for informative markers. Genome-wide association study (GWAS) analysis used a linear model with a transformation y =loge(Pheno1 + 60) applied prior to data analysis and fitted using the lm function in R. From each SNP model fitted, the P value form the F-test was extracted, and adjusted P values (false discovery rate [FDR]) were calculated using the P.adjust function in R. Manhattan plots were constructed with thresholds taken as FDR =0.05 (suggestive) and FDR =0.01 (significant). Back-transformed means for each SNP genotype (AA, AB and BB) along with their standard errors were calculated using the emmeans package in R. For chromosomes where at least one SNP with FDR <0.05 was found, the most significant SNP in the chromosome was identified. In order to assess if any of the other ‘significant SNP’ in the chromosome had any effect in addition to the most significant one, a linear model was fitted with the most significant SNP as well as the SNP to be tested. No formal adjustment for multiple tests was undertaken for this analysis.
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