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Transcriptional Effect of DEFB1 Gene 5' Untranslated Region Polymorphisms

Genetic Service, Instituto di Ricovero e Cura a Carattere Scientifico Burlo Garofolo, Trieste, Italy

To the Editor: Human ß-defensin-1 (hBD-1) has been shown to be a candidate tumor suppressor gene by Sun et al. (1) who reported that single nucleotide polymorphisms (SNP) in the DEFB1 gene are able to modify the transcriptional activity of hBD-1 promoter. In particular, the –44C/G SNP was described to enhance transcription up to 2.3 times more than the wild-type sequence in DU145 or TSU-Pr1 cell lines.

Recently, we showed that significant correlation exists between SNPs in the 5' untranslated region (UTR) of the DEFB1 gene and the risk of being infected with HIV-1 in Italian and Brazilian children (2, 3).

With the aim of verifying an eventual transcriptional functional effect of the SNPs in the 5'UTR region of DEFB1 gene, we employed a dual luciferase assay, the same technique used by Sun et al. (1).

Starting from the wild-type DEFB1 5'UTR region, we generated three mutated inserts using primers designed to introduce the desired mutation into the wild-type sequence. The four different fragments (wild-type, –52A, –44G, and –20A) have been cloned into pGL3 promoter vector (Promega), and Caco2 cells were transfected for 48 h with 1 µg of the reporter plasmids and 20 ng of control Renilla luciferase expression plasmid (phRG-TK, Promega) using 5 µL of GenePORTER transfection reagent; a dual luciferase assay (Promega) was done according to the manufacturer's instruction. To ensure the reproducibility of the results, all tests have been done in quadruplicate and repeated at a later time to confirm the obtained results.

Our results show that the three SNPs do possess a functional activity leading to an impaired production of the gene downstream to the mutated promoter.¹ Both the –52A and the –20A alleles cause a 25% mean reduction of expression. Although quantitatively relevant, this reduction was not statistically significant. The –44G mutation, contrary to the data presented by Sun et al. (1), drag to a markedly reduced expression of about 53% in our experimental setup.

Based on our results (2, 3) and because all the studied SNPs could hamper an effective production of hBD-1 in vivo, we hypothesized that they could be related to reduced response from the innate immune system.

Despite the fact that DU145, TSU-Pr1, and Caco2 are all carcinoma-derived cell lines, their inherent differences could lead to variations in dual luciferase assay results: we believe that the functional effect of the DEFB1 5'UTR SNPs is still controversial and deserves a more comprehensive study.

Footnotes

¹ More detailed information is available at http://www.bbcm.univ.trieste.it/~bbcm/defb1funct/.

References

1. Sun CQ, Arnold R, Fernandez-Golarz C, et al. Human ß-defensin-1, a potential chromosome 8p tumor suppressor: control of transcription and induction of apoptosis in renal cell carcinoma. Cancer Res 2006;66:8542–9.[Abstract/Free Full Text]
2. Braida L, Boniotto M, Pontillo A, Tovo PA, Amoroso A, Crovella S. A single-nucleotide polymorphism in the human ß-defensin 1 gene is associated with HIV-1 infection in Italian children. AIDS 2004;18:1598–600.[CrossRef][Medline]
3. Milanese M, Segat L, Pontillo A, Arraes LC, de Lima Filho JL, Crovella S. DEFB1 gene polymorphisms and increased risk of HIV-1 infection in Brazilian children. AIDS 2006;20:1673–5.[Medline]

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