Development of a high-resolution melting method for the screening of TNFAIP3 gene mutations.

Tumor necrosis factor, α-induced protein 3 (TNFAIP3) which encodes a ubiquitin-modifying enzyme (A20), acts as a negative regulator of the NF-κB pathway, and in lymphoma and autoimmune diseases it is frequently inactivated by mutations and/or deletions. We investigated the prevalence of the inactivation of TNFAIP3 in oral squamous cell carcinoma (OSCC). DNA was extracted from 81 cases of OSCC and 50 peripheral blood samples from normal controls. A high-resolution melting (HRM) analysis was used to characterize TNFAIP3 mutations, and the results were confirmed by direct DNA sequencing. Three mutations and three single-nucleotide polymorphisms (SNPs) were found to be associated with OSCC; the TNFAIP3 mutation occurred in 3.7% (3/81) of the OSCC cases examined. All mutations were in exon 7 [c.1081G>A (p.E361K), c.1398C>G (p.S466R) (rs200878487) and c.1760C>T (p.P587L) (rs150056192)], and p.E361K was identified as a novel mutation. We further used SIFT and PolyPhen-2 software to assess potentially functional mutations. Two SNPs, c.296­20_296-18delCTC (rs71670547) and c.380T>G (p.F127C) (rs2230926), were located in exon 3, and c.2140C>T (p.P714S) was located in exon 9. A novel SNP, p.P714S differed from the one reported previously (p.P714A) (rs369155845) at that site. We also identified five SNPs in 50 normal Taiwanese individuals, and two of them [c.296­15C>T (rs377482653) and c.305A>G (p.N102S) (rs146534657)] were not found in our OSCC tissue. HRM facilitated the screening of genetic changes. In addition, our results indicate that the prevalence of the TNFAIP3 mutation is low in OSCC.

Mutation analysis of 13 driver genes of colorectal cancer-related pathways in Taiwanese patients.

AIM: To investigate the driver gene mutations associated with colorectal cancer (CRC) in the Taiwanese population. METHODS: In this study, 103 patients with CRC were evaluated. The samples consisted of 66 men and 37 women with a median age of 59 years and an age range of 26-86 years. We used high-resolution melting analysis (HRM) and direct DNA sequencing to characterize the mutations in 13 driver genes of CRC-related pathways. The HRM assays were conducted using the LightCycler® 480 Instrument provided with the software LightCycler® 480 Gene Scanning Software Version 1.5. We also compared the clinicopathological data of CRC patients with the driver gene mutation status. RESULTS: Of the 103 patients evaluated, 73.79% had mutations in one of the 13 driver genes. We discovered 18 novel mutations in APC, MLH1, MSH2, PMS2, SMAD4 and TP53 that have not been previously reported. Additionally, we found 16 de novo mutations in APC, BMPR1A, MLH1, MSH2, MSH6, MUTYH and PMS2 in cancerous tissues previously reported in the dbSNP database; however, these mutations could not be detected in peripheral blood cells. The APC mutation correlates with lymph node metastasis (34.69% vs 12.96%, P = 0.009) and cancer stage (34.78% vs 14.04%, P = 0.013). No association was observed between other driver gene mutations and clinicopathological features. Furthermore, having two or more driver gene mutations correlates with the degree of lymph node metastasis (42.86% vs 24.07%, P = 0.043). CONCLUSION: Our findings confirm the importance of 13 CRC-related pathway driver genes in the development of CRC in Taiwanese patients.

MALAT1 long non-coding RNA is overexpressed in multiple myeloma and may serve as a marker to predict disease progression.

BACKGROUND: The pathogenesis of multiple myeloma involves complex genetic and epigenetic events. This study aimed to investigate the role and clinical relevance of the long non-coding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in multiple myeloma. METHODS: Bone marrow mononuclear cells were collected for analysis. The samples of multiple myeloma were taken from 45 patients at diagnosis, 61 post-treatment, and 18 who relapsed or had progression. Control samples were collected from 20 healthy individuals. Real-time quantitative reverse transcription polymerase chain reactions were performed to evaluate the expression of MALAT1. The clinical relevance of MALAT1 expression was also explored. RESULTS: MALAT1 was overexpressed in the newly diagnosed patients compared with post-treatment patients (mean ∆CT: -5.54 ± 0.16 vs. -3.84 ± 0.09, 3.25-fold change; p < 0.001) and healthy individuals (mean ∆CT: -5.54 ± 0.16 vs. -3.95 ± 0.21, 3.01-fold change; p < 0.001). The expression of MALAT1 strongly correlated with disease status, and the magnitude of change in MALAT1 post-treatment had prognostic relevance. The patients with early progression had a significantly smaller change in MALAT1 after treatment (mean ∆CT change: 1.26 ± 1.06 vs. 2.09 ± 0.79, p = 0.011). A cut-off value of the change in MALAT1 (∆CT change: 1.5) was obtained, and the patients with a greater decrease in MALAT1 (difference in ∆CT >1.5) had significantly longer progression-free survival compared with the patients with a smaller MALAT1 change (24 months vs. 11 months; p = 0.001). For the post-treatment patients, the risk of early progression could be predicted using this cut-off value. CONCLUSIONS: MALAT1 was overexpressed in patients with myeloma and may play a role in its pathogenesis. In addition, MALAT1 may serve as a molecular predictor of early progression.