Somatic mutations in oral squamous cell carcinomas in 98 Japanese patients and their clinical implications.

INTRODUCTION: The somatic mutational profile of oral squamous cell carcinoma (OSCC) among Japanese patients has been less investigated, partly because of the rarity of the tumor. Moreover, previous studies have either used formalin-fixed paraffin-embedded samples or lacked paired normal tissues. We aimed to determine somatic mutations in the exomes of 76 genes, including 50 driver genes of solid cancers and NOTCH-related genes, some of which are previously reported as frequently mutated in head and neck squamous cell carcinoma or OSCC. MATERIALS AND METHODS: We used fresh-frozen tumor/normal-paired samples from 98 treatment-naïve Japanese patients with OSCC and analyzed their correlations with clinicopathological characteristics and survival. RESULTS: We identified 136 exonic mutations, including 78 non-synonymous mutations, 13 synonymous mutations, 22 nonsense mutations, 2 non-frameshift deletions, 11 frameshift deletion, and 5 each of splice-site and frameshift insertions. The most frequently mutated genes were TP53 (36.7%), FAT1 (9.2%), NOTCH1 (8.2%), CDKN2A (7.1%), ZFHX4 (5.1%), CASP8 (4.1%), EP300 (4.1%), and KMT2D (4.1%). We followed up 90 of the 98 patients for 3 years. Among them, TP53 mutation was associated with significantly shorter 3-year disease-free survival. Most of the identified TP53 mutations occurred in the DNA-binding domain and were functionally deleterious. DISCUSSION: Our findings and the mutation spectra can contribute to the development of a therapeutic strategy for Japanese patients with OSCC.

New PCNT candidate missense variant in a patient with oral and maxillofacial osteodysplasia: a case report.

BACKGROUND: Osteodysplasia of the oral and maxillofacial bone is generally accompanied by systemic bone abnormalities (such as short stature, joint contracture) or other systemic abnormalities (such as renal, dermatological, cardiovascular, optic, or hearing disorders). However, it does not always present this way. Recent reports have suggested that genome-wide sequencing is an effective method for identifying rare or new disorders. Here, we performed whole-exome sequencing (WES) in a patient with a unique form of acquired, local osteodysplasia of the oral and maxillofacial region. CASE PRESENTATION: A 46-year-old woman presented to our hospital with the complaint of gradually moving mandibular teeth (for 6 months), changing facial appearance, and acquired osteolysis of the oral and maxillofacial bones, showing mandibular hypoplasia without family history. Upon skeletal examination, there were no abnormal findings outside of the oral and maxillofacial area; the patient had a height of 157 cm and bone mineral density (according to dual energy x-ray absorptiometry) of 90%. Results of blood and urine tests, including evaluation of bone metabolism markers and neurological and cardiovascular examinations, were normal. We performed WES of genomic DNA extracted from the blood of this patient and her mother, who did not have the disease, as a negative control. We identified 83 new missense variants in the patient, not detected in her mother, including a candidate single nucleotide variant in exon 14 of PCNT (pericentrin). Critical homozygous or compound heterozygous variants in PCNT are a known cause of microcephalic osteodysplastic primordial dwarfism type II accompanied by mandibular hypoplasia, which is similar to the maxillofacial phenotype in this patient. CONCLUSIONS: Protein simulations performed using Polymorphism Phenotyping v2 and Combined Annotation Dependent Depletion software indicated that this missense variant is likely to disrupt the PCNT protein structure. These results suggest that this is a new form of osteolysis related to this PCNT variant.

Trousseau syndrome in a patient with advanced oral squamous cell carcinoma: a case report.

BACKGROUND: Trousseau syndrome is known as a variant of cancer-associated thrombosis. Trousseau syndrome commonly occurs in patients with lung or prostate cancer. Hypercoagulability is thought to be initiated by mucins produced by the adenocarcinoma, which react with leukocyte and platelet selectins to form platelet-rich microthrombi. This is the first report of Trousseau syndrome in a patient with oral cancer. CASE PRESENTATION: Here, we describe the case of a 61-year-old Japanese man diagnosed as having advanced buccal carcinoma (T4bN2bM1; the right scapula, erector spinae muscles, and the right femur), who experienced aphasia and loss of consciousness. Although magnetic resonance imaging showed cerebral infarction, carotid invasion by the tumor and carotid sheath rupturing, cardiovascular problems, and bacterial infection were not present, which indicated Trousseau syndrome. CONCLUSIONS: Trousseau syndrome in oral cancer is rare, but we must always consider cancer-associated thrombosis in patients with advanced stages of cancer regardless of the primary site of the cancer and take steps to prevent it.

Enzyme linking to DNA aptamers via a zinc finger as a bridge.

We propose a novel enzyme-labeling method for DNA aptamers using enzyme-fused zinc finger proteins. We achieved thrombin detection and vascular endothelial growth factor detection using zinc finger-fused firefly luciferase.

Detection of pathogenic bacteria by using zinc finger protein fused with firefly luciferase.

We constructed a novel bacterial genome detection system using zinc finger protein (ZF) fused with firefly luciferase (ZF-luciferase). Taking advantage of the direct recognition of double-stranded DNA (dsDNA) by ZF, we previously constructed bacterial genome detection systems that did not require dehybridization processes. To detect polymerase chain reaction (PCR) products rapidly and with a high sensitivity, we constructed two kinds of ZF-luciferase, Sp1-fused luciferase (Sp1-luciferase), and Zif268-fused luciferase (Zif268-luciferase). ZF-luciferase not only maintains luciferase activity but also shows dsDNA-binding ability and specificity. Furthermore, we succeeded in the detection of 10 copies of the genome of Legionella pneumophila and Escherichia coli O157. ZF-luciferase would be a useful tool for highly sensitive detection of pathogenic bacterial genome.

Zn finger-based direct detection system for PCR products of Salmonella spp. and the Influenza A virus.

We have detected PCR products from Salmonella spp. and Influenza A virus using Zn finger protein Zif268 and Sp1, respectively. Previously, we demonstrated a novel method of rapid and specific detection of PCR products from Legionella pneumophila genome using Zn finger protein Sp1. In principle, this methodology might be applied to the detection of most bacteria and viruses using various Zn finger proteins. Here, to demonstrate the wider applicability of our method, we detected PCR products from Salmonella spp. and the Influenza A virus. BLAST data indicated the Zif268 and Sp1 recognition sequence were located on the gyrB gene of Salmonella spp. and the nucleoprotein gene of Influenza A virus, respectively. The PCR products from the oligonucleotide corresponding to the gyrB gene of Salmonella spp. or the nucleoprotein gene of the Influenza A virus could be specifically detected by ELISA or fluorescence depolarization measurement using Zif268 or Sp1. These results indicate the wide applicability of our novel methodology.

Zinc finger protein-based detection system of PCR products for pathogen diagnosis.

A novel detection system of PCR products from bacterial genomes using Zinc finger proteins was developed. Zinc finger proteins are DNA-binding proteins that can bind to dsDNA with high affinity and specificity. Since Zinc finger proteins can directly detect PCR products and can double-check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to construct the detection system for three pathogen, Legionella pneumophila, Salmonella spp. and Influenza A virus using well-characterized Zinc finger proteins. As a result, we succeeded in detecting the PCR products from Legionella pneumophila, Salmonella spp. and Influenza A virus using Sp1 and Zif268. Therefore, this methodology can be applied to the detection of most pathogen using various Zinc finger proteins.

The simple and rapid detection of specific PCR products from bacterial genomes using Zn finger proteins.

A novel method of rapid and specific detection of polymerase chain reaction (PCR) products from bacterial genomes using Zn finger proteins was developed. Zn finger proteins are DNA-binding proteins that can sequence specifically recognize PCR products. Since Zn finger proteins can directly detect PCR products without undergoing dehybridization, unlike probe DNA, and can double check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to detect Legionella pneumophila using Sp1. It was found that a 49 bp L. pneumophila-specific region containing the Sp1 recognition site is located on the flhA gene of the L. pneumophila genome. We succeeded in specifically detecting PCR products amplified from L. pneumophila in the presence of other bacterial genomes by ELISA, and demonstrated that Sp1 enables the discrimination of L. pneumophila-specific PCR products from others. By fluorescence depolarization measurement, these specific PCR products could be detected within 1 min. These results indicate that the rapid and simple detection of PCR products specific to L. pneumophila using a Zn finger protein was achieved. This methodology can be applied to the detection of other bacteria using various Zn finger proteins that have already been reported.

Specific detection of PCR product from Legionella pneumophila strain Philadelphia1 using zinc finger protein Sp2.

We have developed a novel method to detect PCR products from pathogen genome DNA by Zinc finger protein that can bind to double strand DNA (dsDNA) in sequence specific manner. In this study, we tried to detect Legionella pneumophila strain Philadelphia 1 using Zinc finger protein. We found the specific target DNA sequence for zinc finger protein Sp2 in L. pneumophila strain Philadelphia 1 genome DNA. Specific PCR product was successfully amplified from L. pneumophila strain Philadelphia 1 genome DNA and we used Zinc finger protein Sp2 to detect it. We succeeded in detecting the PCR products from L. pneumophila strain Philadelphia 1 genome DNA with Sp2.