**5. Conclusions**

We showed that the great variability among 5′UTR features seen on a whole genome level can be observed even in the group of homologous ABCA subfamily genes. Our phylogenetic analysis, based on the comparison of nucleotide sequences of ABCA 5′UTRs, shows that the 5′UTRs of *ABCA2* and *ABCA3* cluster distinctly from the rest of the 5′UTRs. The 5′UTRs of ABCA genes contain above-average numbers of uATGs, uORFs and 5′UTR introns as well as conserved ones and these elements probably play important biological roles in this subfamily, unlike RG4s. Our work brings largely new information on the numbers of stem loops in 5′UTRs. Some of the positive correlations among 5′UTR features are likely, however, some are interesting and need further exploration, such as the correlations between number of uATGs and number of 5′UTR introns or sATG flanking sequence context and presence of RG4-forming sequence. The lengths of the ABCA 5′UTRs seem to be the major factor influencing the numbers of the other known 5′UTR regulatory elements. Although there is also great variability in the distribution and expression of the ABCA proteins, we did not find any significant correlation between the numbers of 5′UTR features and protein expression characteristics. However, we confirmed a high concentrations of ribosomes at some of the predicted uORFs in the analysis of Ribo-seq data. We further verified the existence of SNVs in relation to the 5′UTR features, predicted within this study, experimentally in our cohort of 105 breast cancer patients. Our results support the view that the other elements such as RNA binding proteins and non-coding RNAs play the major role in protein expression fine-tuning within the complex background of the highly variable 5 ′UTRs. These findings extend our view on human genome variability and raise new questions for further investigations.

**Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/22/ 8878/s1. **Table S1.** Basic characteristics of the transcripts downloaded and analyzed in the study. **Table S2.** A detailed overview of 5′UTR features in the human ABCA genes. **Table S3.** Correlation tables of the first correlation analysis with Spearman's rs and Kendall's tau coefficients and relevant p values. **Table S4.** Correlation tables of the second correlation analysis with Spearman's rs and Kendall's tau coefficients and relevant p values. **Figure S1.** Multi-sequence alignment (Clustal Omega algorithm) of the 5′UTRs of the human ABCA1 gene and its vertebrate orthologs with nucleotide percentage identity colored, consensus logos and occupancy score histograms. **Figure S2.** Multi-sequence alignment (Mafft algorithm) of the 5′UTRs of the human ABCA1 gene and its vertebrate orthologs with nucleotide percentage identity colored, consensus logos and occupancy score histograms. **Figure S3.** Multi-sequence alignment (Clustal Omega algorithm) of the 5′UTRs of all 12 human ABCA protein-coding genes with nucleotide percentage identity colored, consensus logos and occupancy score histograms. **Figure S4.** Multi-sequence alignment (Mafft algorithm) of the 5′UTRs of all 12 human ABCA protein-coding genes with nucleotide percentage identity colored, consensus logos and occupancy score histograms. **Figure S5.** The location of 13 SNVs, found in breast cancer patients, in relation to the 5′UTR features of ABCA genes.

**Author Contributions:** Conceptualization: P.D.; methodology: P.D.; software: P.D.; validation: P.D., V.H., and P.S.; formal analysis: P.D.; investigation: P.D., V.H., and P.S.; resources: P.D., V.H., and P.S.; data curation: P.D.; writing—original draft preparation: P.D.; writing—review and editing: P.D., V.H., and P.S.; visualization: P.D.; supervision: P.S.; project administration: P.D., V.H., and P.S.; funding acquisition: P.D., V.H., and P.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic project INTER-COST no. LTC19015 (to P.S.), project no. CZ.02.1.01/0.0/0.0/16\_019/0000787 "Fighting INfectious Diseases", awarded by the MEYS CR, financed from EFRR (to P.D.) and Charles University project "Center of clinical and experimental liver surgery" no. UNCE/MED/006 (to V.H.).

**Acknowledgments:** We would like to thank Sarah Leupen, PhD. from the University of Maryland Baltimore County for English language review.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
