Heirarchical cluster analysis of 14 gender-specific genes as a novel validation of microarray expression data. Genes were chosen on the basis of comparison of mean gene expression levels between 8 male and 6 female sickle cell disease patients (HbSS phenotype only, designated as either M-SS for males or F-SS for females) with false discovery rate of less than or equal to 10%. Hierarchical cluster analysis of the expression pattern of these 16 probe sets (14 genes) in all sickle cell disease patients (HbSS phenotype), including the original 14 subjects as well as 10 additional sickle cell disease patients on hydroxyurea (M-SS* or F-SS*) and 13 controls (M-AA or F-AA) show that they discriminate gender with 100% accuracy. In this figure each column represents a sickle cell disease patient or a control subject and each row represents a probe set. Red signifies increased expression and green signifies decreased expression. Genes that are up-regulated in males over females are generally located on the Y chromosome and genes that are up-regulated in females over males are generally located on the X chromosome. One gene, plakophilin 2, maps to an autosomal location and has a low overall expression level. With the selected false discovery rate of less than or equal to 10% one would only expect 1 or 2 false positives. Eukaryotic translation initiation factor 1A is represented by 3 different probe sets. All 4 X-linked genes (underlined and highlighted in yellow) represent X chromosome genes known to escape X inactivation. This specificity is a remarkable additional validation of the experimental and analytical methodology.