is a differentially expressed gene in Black versus White HKCs and HNSCCs of prognostic significance.Figure 4(A) Differentially expressed genes (DEGs) in Black versus White HKC strains identified in the present study were compared with those in a publicly available data set of HNSCCs (TCGA Firehose Legacy, 520 patients) selected on the basis of the same criteria (|log2(Foldchange)|>0.5 and P value <0.05) as for analysis of the HKC profiles in Table S2. Shown is the list of the overlapping DEGs together with a heat map indication of average log2 Fold difference and corresponding statistical significance in both HKC and TCGA (HNSCC) data sets (within brackets). (B) Kaplan Meier plots of long-term overall survival of H/NSCC patients from the TCGA dataset divided on the basis of high versus low HSD17B7 expression levels relative to its median expression value among males (darkgreen and lightgreen lines) and females (red and orange lines), showing statistically significant association of elevated HSD17B7 expression with poorer patient survival. Statistical significance was calculated by the log-rank test between the groups. High_female: n=57patients. Low_female: n= 79 patients. High_male: n= 203 patients. Low_male: n= 181 patients. (C) Single and multiple COX regression analysis of HNSCC data sets from the TCGA for high risk (HR) of patients' death as a function of levels of HSD17B7 expression, either as a single variable or after adjusting for gender, ethnicity, age or all three together. Only HNSCC samples from patients of White (Caucasian) versus Black ancestries were used for this analysis. Cox proportional hazards regression model was fitted with the R function coxph. (D) Gene Ontology (GO) analysis of the list of genes that positively correlate with HSD17B7 expression in HKC strains and HNSCCs, with corresponding Benjamini scores (left and right columns, respectively). GO categories are divided into Biological Processes and Cellular Components and the most significant categories in both HKC strains and TCGA (HNSCC) are reported. (E) Correlation analysis between the holoclone forming capability of HKC strains (as determined in Fig 2E) and HSD17B7 expression levels in the corresponding transcriptomic profiles. Values for individual HKC strains are indicated by dots. n(HKC strains)=44. The linear regression R (0.53) and p-value (0.0004) are indicated. The graph shows absolute expression values (probe intensity after global normalization by TAC software) obtained from human Clariom D microarrays. The statistical test is a simple linear regression. (F) RT-qPCR analysis of HSD17B7 expression in the indicated HKC strains derived from White versus Black donors (White and Black bars), as a validation of the corresponding transcriptomic profiles of Fig 3A.**p<0.01. (G) Immunofluorescence (IF) analysis of HSD17B7 protein expression in White and Black HKCs. Shown are representative images as well as IF signal quantification at the level of individual cells (dots) for the indicated strains. 19-46 cells were counted in each case. n(HKC strains per ancestry) = 5, ***p<0.0005, horizontal line: median, 2-tailed unpaired t-test. Unit of Y-axis: pixels. Scale bar: 20 μm. (H) Map of eQTLs position within a 1 MB region encompassing the HSD17B7 gene, with corresponding exons shown as high blue bars, and the additional indicated genes. Shown are all eQTLs associated with HSD17B7 expression in the HKC transcriptomic profiles, with the ones with differential distribution in the Black versus White HKC strains indicated in colors. Position of the eQTLs affecting recognition of the indicated transcription factors is also shown. (I) Plots with allele frequency and associated levels of HSD17B7 expression in Black versus White samples for two of the identified eQTLs (rs4542196 and rs10737487). Log2 Intensity values are reported on the y axis, while genotypes (AA, GA, GG) are reported on the x axis. Rs4542196 (AA: 19 White, 2 Black; GA: 3 White, 14 Black; GG: 1 White, 12 Black). Rs10737487 (AA: 18 White, 1 Black; GA: 5 White, 15 Black; GG: 1 White, 12 Black). Boxes represent the first (lower) quartile, the median (central band) and the third (upper) quartile. Whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box. (J) Summary table of eQTLs with statistically significant association with HSD17B7 expression in experimentally determined HKC transcription profiles (52 strains) and differential distribution in Black versus White populations by analysis of 1000 Genomes Phase I May 2011 (AFRICA (N=246), EUROPE (N=380)) with Fixation index (Fst ) value >0.3. The effect of the genotype was modeled as additive linear and tested for its significance (p-values) using t-statistic. (K) Linkage analysis on the six reported SNPs using the LDmatrix tool (https://ldlink.nci.nih.gov/?tab=ldmatrix). Pairwise linkage disequilibrium statistics, calculated on all populations (AFR, AMR, EAS, EUR, SAS), are reported as heat maps of shaded colors corresponding to the indicated gradient of values (red: R2; blue: D'). The specific R2 and D' numbers are provided in Appendix Table S3.