Scram1 is a modifier of spinal cord resistance for astrocytoma on mouse Chr 5

Abstract

Tumor location can profoundly affect morbidity and patient prognosis, even for the same tumor type. Very little is known about whether tumor location is determined stochastically or whether genetic risk factors can affect where tumors arise within an organ system. We have taken advantage of the Nf1-/+;Trp53-/+cis mouse model of astrocytoma/glioblastoma to map genetic loci affecting whether astrocytomas are found in the spinal cord. We identify a locus on distal Chr 5, termed Scram1 for spinal cord resistance to astrocytoma modifier 1, with a LOD score of 5.0 and a genome-wide significance of P < 0.004. Mice heterozygous for C57BL/6J×129S4/SvJae at this locus show less astrocytoma in the spinal cord compared to 129S4/SvJae homozygous mice, although we have shown previously that 129S4/SvJae mice are more resistant to astrocytoma than C57BL/6J. Furthermore, the astrocytomas that are found in the spinal cord of Scram1 heterozygous mice arise in older mice. Because spinal cord astrocytomas are very rare and difficult to treat, a better understanding of the genetic factors that govern astrocytoma in the spine may lead to new targets of therapy or prevention.

Figure 1

Spinal cord GBM in a BC(129XB6)X129-NPcis mouse. Panel A shows a cross-section view at low magnification with the GBM (arrows) diffusely infiltrating one side of the spinal cord. The high magnification view (B) shows densely packed irregular oblong nuclei and a region of hemorrhage (arrows). Panel C shows low-magnification, of a longitudinal section of the same tumor, highlighting the widespread extent of the tumor in the spinal cord with hemorrhage (arrows) and regions of necrosis (arrowheads). The high magnification view (D) shows a necrotic region (NR) with weakly pallisading tumor cells (arrows), blood vessels (BV) with evidence of microvascular proliferation, and atypical mitoses (arrowheads).

Figure 2

Binary trait linkage analysis of spinal cord astrocytoma in BC(129XB6)X129)-NPcis mice. Panel A shows the linkage across the genome, with a peak on Chr 5. The dotted line shows the genome-wide significance level of P = 0.05 based on permutation testing. Panel B shows linkage on Chr 5 with the highest peak at 77.26 cM. The horizontal bracket below the peak indicates the 1.5 LOD support interval that defines the Scram1 locus. The rs4225536 marker that was used for subsequent analyses is indicated by the arrow at 75.7 cM.

Figure 3

The B6 allele at Scram1 affects both the overall levels of astrocytoma and the ratio of astrocytomas found in the spinal cord in backcross mice. The incidence of spinal cord astrocytoma compared to all astrocytoma is shown at marker rs4225536, nearest to the Scram1 peak. Statistically significant differences were found between the incidence of total astrocytoma in the two groups (Fisher exact test P = 0.013), the incidence of spinal cord astrocytoma in the two groups (Fisher exact test P <0.0001), and the ratios of spinal cord astrocytoma to total astrocytoma in the two groups (Fisher exact test P = 0.0002). The upward error bar indicates the 95% CI for all astrocytomas and the downward error bar indicates the 95% CI for spinal cord astrocytomas (dark gray). SS indicates the 129/129 genotype at rs4225536 and SB indicates the 129/B6 genotype.

Figure 4

Scram1 modifies timing of astrocytoma in the spinal cord, but not astrocytoma overall. Samples were stratified by the rs4225536 marker nearest the Scram1 peak. Spinal cord astrocytoma-free survival is delayed in Scram1^129/B6 (SB) mice compare to Scram1^129/129 (SS) mice (A). Astrocytomas overall are observed at the same ages in Scram1^129/B6 (SB) and Scram1^129/129 (SS) mice (B). P values for Kaplan-Meier analysis are given in each panel. N.S. = not significant. Median survival age is graphed for mice specifically with spinal cord astrocytomas and for mice with astrocytomas anywhere in the central nervous system (C) demonstrating that Scram1^129/B6 mice have a delay in the presentation of astrocytoma in the spinal cord.

Figure 5

Synteny of human chromosomes to Scram1. The top cyan bar represents the mouse Scram1 locus. Human chromosome segments are shown below, with the Chr 12 segment in red and Chr 7 segments in shades of blue. The Mb coordinates of each segment are shown at the bottom of the diagram. The bar graph shows amplification (red) and deletion (blue) of these regions in human brain astrocytoma samples taken from the REMBRANDT Astrocytoma Project and visualized in the Cancer Workbench Heatmap Viewer (https://cgwb.nci.nih.gov/cgi-bin/heatmap). Vertical black lines show the division between the different chromosome segments aligned to mouse Scram1 and horizontal gray lines indicate amplification or deletion in 20% and 40% of samples. Genes within the regions of most frequent amplification are indicated, with the genes that are polymorphic between B6 and 129 in red type.

Figure 6

Ratio of B6 expression levels to 129/SvImJ expression levels of Scram1 genes in normal male mouse brains. Publically available gene expression data (http://phenogen.ucdenver.edu/PhenoGen/index.jsp) was analyzed for genes in the Scram1 region that had ≥ 1.5-fold difference in expression level between B6 and 129/SvImJ. Only genes that met this threshold are shown. Red bars indicate genes that are more highly expressed in B6 compared to 129/SvImJ and blue bars indicate genes that are higher in 129/SvImJ. Gene symbols are listed in alphabetical order along the Y-axis.