EWS/FLI-responsive GGAA microsatellites exhibit polymorphic differences between European and African populations

Abstract

The genetics of Ewing sarcoma development remain obscure. The incidence of Ewing sarcoma is ten-fold less in Africans as compared to Europeans, irrespective of geographic location, suggesting population-specific genetic influences. Since GGAA-containing microsatellites within key target genes are necessary for Ewing sarcoma-specific EWS/FLI DNA binding and gene activation, and gene expression is positively correlated with the number of repeat motifs in the promoter/enhancer region, we sought to determine if significant polymorphisms exist between African and European populations which might contribute to observed differences in Ewing sarcoma incidence and outcomes. GGAA microsatellites upstream of two critical EWS/FLI target genes, NR0B1 and CAV1, were sequenced from subjects of European and African descent. While the characteristics of the CAV1 promoter microsatellites were similar across both populations, the NR0B1 microsatellite in African subjects was significantly larger, harboring more repeat motifs, a greater number of repeat segments, and longer consecutive repeats, than in European subjects. These results are biologically intriguing as NR0B1 was the most highly enriched EWS/FLI bound gene in prior studies, and is absolutely necessary for oncogenic transformation in Ewing sarcoma. These data suggest that GGAA microsatellite polymorphisms in the NR0B1 gene might influence disease susceptibility and prognosis in Ewing sarcoma in unanticipated ways.

Figure 1

NR0B1 and CAV1 GGAA-microsatellite sequence characteristics are visualized in panels A and B. The NR0B1 and CAV1 microsatellites are located −1.6 to −1.1kb and −2.0 to −1.7kb upstream from the transcription start site (TSS), respectively in the promoter/enhancer region. Using customized computer software, microsatellite sequences and pre-defined microsatellite characteristics were identified and quantified from the raw sequencing files. The NR0B1 GGAA-microsatellite is characterized by repeat segments separated by a single adenosine nucleotide, whereas the CAV1 GGAA-microsatellite is characterized by numerous repeat segments partitioned by a variable number of AGAA repeat motifs.

Figure 2

Histogram plots demonstrating the distribution of the CAV1 GGAA microsatellite characteristics in European and African populations. The data plots for the total number of repeats (Panel A), the total microsatellite length (Panel C) and the number of repeat segments (Panel D) show very similar distributions comparing European and African populations.

Figure 3

Histogram plots demonstrating the distribution of the NR0B1 GGAA microsatellite characteristics in European and African populations. Panel A illustrates the total number of repeats, showing a predominant clustering of the European data around shorter sequences consisting of 16-25 repeats, whereas the African data is much more disperse with a greater distribution towards larger repeat numbers. Similar trends are depicted for the total microsatellite length (Panel C) and the number of repeat segments (Panel D).

Figure 4

Stratification of the NR0B1 microsatellite data illustrating the proportion of European and African microsatellite sequences above and below the modal distribution of the total number of GGAA repeats (panel A) and the longest consecutive repeat sequence (panel B).