|Functional Genomics Section
DOE Human Genome Program Contractor-Grantee Workshop
140. Genetic Factors Affecting Globin Switching
Sluan D. Lin, Phil Cooper, Mary
E. Stevens, and Edward M. Rubin
Low level expression of fetal gamma globin inhibits red cell sicklying and its pathological consequence in individuals homozygous for the Beta-S alleles. However, fetal gamma globin switches adult beta globin shortly after birth. To furthering our understanding of the genetic factors affecting the sickle cell disease, we are studying the "transacting" modifier gene(s) that impact on the switching of human gamma to beta globin in transgenic mice.
Creation of transgenic mice that persistently express human gamma globin: We have made transgenic mice using a YAC containing the entire human beta-cluster with a -117 Agm mutation. The mutation causes the Greek form of hereditary persistence of fetal hemoglobin (HPFH) in human. We found that the HPFH mutation can also causes the human gamma chains to be expressed postnatally in the transgenic mice. This feature has greatly facilitated the study of the globin switching parameters and the level of gamma globin expression after birth.
Different genetic backgrounds affect the globin switching parameter among the F1 animals: The heterozygous FVB transgenic animals were crossed with different inbred strains including DBA/2N, Balb/C, 129/SvJ and SWR/J. The blood of YAC-positive animals screened by PCR were collected on 10, 15, 30 and 60 days after birth. The human gamma/beta globin ratio of the DBA/2N-derived F1 hybrid shows consistent highest level throughout the sampling period. Comparing DBA/2N-derived F1 with the transgenic FVB, the p-values are 1x10-7 and 3x10-10 on day 30 and day 60, respectively. Thus, we've verified the hypothesis that different genetic background of F1 hybrid mice, derived from crossing the transgenic with other inbred strains, can affect the level of gamma globin expression.
Backcross suggests more than one genetic loci contribute in regulating gamma globin expression: We first generated 70 backcross transgenic animals as a pilot study of the possible number of genetic factors involved in up-regulating the gamma expression. The lack of a bimodal distribution of the backcross animals suggesting that there are more than one genetic loci that contribute in regulating the expression of human gamma globin in the transgenic mice. By applying the classical formula of Wright (1968), we estimate the number of QTLs controlling the gamma expression is 2.4 and the genetic contribution to the phenotypic variance is 48%. By comparing to others publication of similar situation, We estimate that we would need approximately 200 backcross transgenic animals in total to map the QTLs. To test the polymorphism between the FVB and DBA/2N genomic DNA, we are screening for 83 SSLP markers across the mouse genome at an average genetic interval of 17 CM. Using these markers, we are performing a genome scan starting with the 20% backcross animals at the two phenotypic extremes. Once a significant lod score of 3.3 is achieved, we will pick more markers around the particular locus to fine map the modifier gene.
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