Chimeric Negative Regulation ofp14ARFandTBX1by a t(9;22) Translocation Associated with Melanoma, Deafness, and DNA Repair Deficiency

Abstract

Melanoma is the most deadly form of skin cancer and DiGeorge syndrome (DGS) is the most frequent interstitial deletion syndrome. We characterized a novel balanced t(9;22)(p21;q11.2) translocation in a patient with melanoma, DNA repair deficiency, and features of DiGeorge syndrome (DGS) including deafness and malformed inner ears. Using chromosome sorting we located the 9p21 breakpoint in CDKN2A intron 1. This resulted in under-expression of the tumor suppressor p14ARF; the reduced DNA repair was corrected by transfection with p14ARF. UV-type p14ARF mutations in his melanoma implicated p14ARF in its pathogenesis. The 22q11.2 breakpoint was located in a palindromic AT-rich repeat (PATRR22). We identified a new gene, FAM230A that contains PATRR22 within an intron. The 22q11.2 breakpoint was located 800 kb centromeric to TBX1, which is required for inner ear development. TBX1 expression was greatly reduced. The translocation resulted in a chimeric transcript encoding portions of p14ARF and FAM230A. Inhibition of chimeric p14ARF-FAM230A expression increased p14ARF and TBX1 expression and improved DNA repair. Expression of the chimera in normal cells produced dominant negative inhibition of p14ARF. Similar chimeric mRNAs may mediate haploinsufficiency in DGS or dominant negative inhibition of other genes such as those involved in melanoma.

Clinical Significance of a Point Mutation in DNA Polymerase Beta (POLB) Gene in Gastric Cancer

“…Protein extraction and Western blot analysis with chemiluminescent detection were as described 19 . The following antibodies and dilution factors were used: affinity purified goat polyclonal antibody POLB (sc-5927, Santa Cruz, 1:800), rabbit anti-actin (H-196, Santa Cruz, 1:1000), donkey anti-goat IgG-HRP (sc-2020, Santa Cruz, 1:2000) and goat anti-rabbit IgG-HRP (sc-2030, Santa Cruz, 1:2000).…”

“…Primer sequences are available upon request. Real-time qRT-PCR assays were carried out on a Bio-Rad iCycler iQ system (Bio-Rad, Hercules, CA, USA) using SYBR Green reagent as described 19 .…”

“…Cells were seeded at 2 × 10 4 cells per well on glass coverslips in six-well plates and fixed in 2% paraformaldehyde as described previously 19 . Confocal images were obtained using a LSM 510 Confocal microscope (Carl Zeiss).…”

“…DNA repair capability of cells with wild-type and T889C mutant POLB were assessed using post-UV HCR as described previously 19 20 . pCMVLuc reporter gene plasmid (a kind gift from Dr. Kenneth H. Kraemer, National Cancer Institute ) was dissolved in 10 mm Tris-HCl, 1 mm EDTA, pH 8 (TE buffer) to a final concentration of 100 μ g/ml and poured in a Petri dish to form 1D 2 mm thick layer.…”

Analysis of the t(3;8) of hereditary renal cell carcinoma: a palindrome-mediated translocation

“…These translocations have been reported in unrelated families and the breakpoints in almost all translocation carriers are localized at the center of the PATRR on both chromosomes (10–15). Similarly, a t(1;22)(q21;q11), t(9;22)(p21;q11) and a t(4;22)(q35;q11), all non-recurrent translocations, were reported as PATRR mediated (16–18). To date, all PATRR-mediated translocations have involved the PATRR on chromosome 22 (PATRR22).…”