Genotyping of
ELOVL4 promoter in the whole family components highlighted that the proband's mother and father present an alternate heterozygosity for each variant in exam (
Fig. 4), indicating that the probandinherited both variants in trans. The predictive analysis of the proband's
ELOVL4 promoter by TRANSFAC Professional evidenced the loss of one group of TF binding sites (ETF, ZF5, E2F-6, FBI-I, HDAC2, and TAFII250) in the double heterozygous genotype, due to the presence of c.-90 G>C for most of them. The exception was represented by FBI-I and the complement 666 through 679 binding sites for TAFII250, whose loss was attributable to c.-236 C>T. A second group analysis, resulting from the combined predictions of TRANSFAC and PROMO, revealed the appearance of new possible binding sites of different TFs (CPB, BCL6B secondary motif, Spi-B, Pax-4, RXR-alpha, GKLF, POLR3A, TFII-I, Pax-5, p53, SP1, and GR-alpha), determined by c.-236 C>T for Spi-B, Pax-4 and RXR-alpha, and by c.-90 G>C for others (for further details see
Table 1). This data suggest a probable transcription variation, due to the altered balance of TF binding properties. Furthermore, it is important to understand the relationship between the analyzed TFs, and how each one could influence the others. This was examined by Cytoscape pathway analysis, along with its MCODE plug-in, from which arose a 4-cluster division that highlighted a relevant network involving most of the TFs in exam (
Table 2;
Fig. 5). These data were confirmed by Dual-Luciferase Reporter Assay, involving the proband's
ELOVL4 promoter, compared in its wild form, versus both variants and c.-90 G>C and c.-236 C>T only samples. Results showed an expression reduction of approximately 14% in the c.-90 G>C sample and of approximately 18% in the c.-236 C>T one (compared with a healthy control), but a strong decrease (∼97%) arose from the promoter carrying the combination of above variants (
Fig. 6). The 1-way ANOVA, after Bonferroni's correction, confirmed the statistical significance of analysis (
P < 0.05). Multiple comparison details are listed in
Supplementary Materials.