Kunihiro Fukushima

Genomic structure of the human ING1 gene and tumor-specific mutations detected in head and neck squamous cell carcinomas

We characterized the genomic structure of the human ING1 gene, a candidate tumor suppressor gene, and found that the gene has three exons. We also demonstrated that four mRNA variants were transcribed from three different promoter regions. Of 34 informative cases of head and neck squamous cell carcinoma, 68% of tumors showed loss of heterozygosity at chromosome 13q33-34, where the ING1 gene is located. Here we present the first report that three missense mutations and three silent changes were detected in the ING1 gene in 6 of 23 tumors with allelic loss at the 13q33-34 region. These missense mutations were found within the PHD finger domain and nuclear localization motif in ING1 protein, probably abrogating the normal function.

Allelic loss and reduced expression of the ING3, a candidate tumor suppressor gene at 7q31, in human head and neck cancers

Loss of heterozygosity (LOH) has been frequently detected at chromosome 7q31 region in human head and neck squamous cell carcinomas (HNSCC) and many other cancers, suggesting the existence of tumor suppressor genes (TSG). We analysed LOH at 7q31 region in 49 HNSCC by using six polymorphic microsatellite markers and found allelic deletion in 48% (22/46) of the informative cases. We detected two preferentially deleted regions, one is around D7S643 and the other around D7S486. When we redefined the map of 7q31 region according to the contiguous sequences, a recently identified gene, ING3, was found in the proximity of D7S643. ING3 protein harbors the PHD domain highly homologous among ING family proteins, in which we previously found mutations in a related gene, ING1. As only one missense mutation of the ING3 gene was found in HNSCC, we examined the expression level. Reverse-transcription-PCR analysis demonstrated decreased or no expression of ING3 mRNA in 50% of primary tumors as compared with that of matched normal samples. Especially, about 63% of tongue and larynx tumors showed the decrease and a tendency of higher mortality was observed in cases with decreased ING3 expression. All these findings suggest a possibility that the ING3 gene functions as a TSG in a subset of HNSCC.

Genome-Wide Analyses on Loss of Heterozygosity in Head and Neck Squamous Cell Carcinomas

Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor survival rate. Identifying the tumor suppressor gene (TSG) loci by genomic studies is an important step to uncover the molecular mechanisms involved in HNSCC pathogenesis. We therefore performed comprehensive analyses on loss of heterozygosity (LOH) using a genome-wide panel of 191 microsatellite markers in 22 HNSCC samples. We found 53 markers with significantly high LOH (>30%) on 21 chromosomal arms; the highest values of those were observed on 3p, 9p, 13q, 15q, and 17p, corresponding to D3S2432 (67%), D9S921-D9S925 (67%) and GATA62F03 (86%), D13S1493 (60%), D15S211 (62%), and D17S1353 (88%), respectively. Fifteen hot spots of LOH were defined in 13 chromosomal arms: 2q22-23, 4p15.2, 4q24-25, 5q31, 8p23, 9p23-24, 9q31.3, 9q34.2, 10q21, 11q21-22.3, 14q11-13, 14q22.3, 17p13, 18q11, and 19q12 as loci reported previously in HNSCCs. Furthermore, we identified five novel hot spots of LOH on three chromosomal arms in HNSCC at 2q33 (D2S1384), 2q37 (D2S125), 8q12-13 (D8S1136), 8q24 (D8S1128), and 15q21 (D15S211). In conclusion, our comprehensive allelotype analyses have unveiled and confirmed a total of 20 possible TSG loci that could be involved in the development of HNSCC. These results provide useful clues for identification of putative TSGs involved in HNSCC by fine mapping of the suspected regions and subsequent analysis for functional genes.

Expression and mutation analysis of epidermal growth factor receptor in head and neck squamous cell carcinoma.

The epidermal growth factor receptor (EGFR)–RAS–RAF–mitogen‐activated protein kinase signaling cascade is an important pathway in cancer development and recent reports show that EGFR and its downstream signaling molecules are mutated in a number of cancers. We have analyzed 91 Japanese head and neck squamous cell carcinomas (HNSCC) and 12 HNSCC cell lines for mutations in EGFR, ErbB2, and K‐ras. Exons encoding the hot‐spot regions in the tyrosine kinase domain of both EGFR (exons 18, 19, and 21) and ErbB2 (exons 18–23), as well as exons 1 and 2 of K‐ras were amplified by polymerase chain reaction and sequenced directly. EGFR expression was also analyzed in 65 HNSCC patients using immunohistochemistry. Only one silent mutation, C836T, was found in exon 21 of EGFR in the UT‐SCC‐16A cell line and its corresponding metastasic cell line UT‐SCC‐16B. No other mutation was found in EGFR, ErbB2, or K‐ras. All tumors showed EGFR expression. In 21 (32%) tumors, EGFR was expressed weakly (+1). In 27 (42%) tumors it was expressed (+2) moderately, and in 17 (26%) tumors high expression (+3) was detected. Overexpression (+2, +3) was found in 44 tumors (68%). A worse tumor differentiation and a positive nodal stage were significantly associated with EGFR overexpression (P = 0.02, P = 0.032, respectively). Similar to patients from western ethnicity, mutations are absent or rare in Japanese HNSCC. Protein overexpression rather than mutation might be responsible for activation of the EGFR pathway in HNSCC. (Cancer Sci 2008; 99: 1589–1594)

Massively Parallel DNA Sequencing Successfully Identifies New Causative Mutations in Deafness Genes in Patients with Cochlear Implantation and EAS

Genetic factors, the most common etiology in severe to profound hearing loss, are one of the key determinants of Cochlear Implantation (CI) and Electric Acoustic Stimulation (EAS) outcomes. Satisfactory auditory performance after receiving a CI/EAS in patients with certain deafness gene mutations indicates that genetic testing would be helpful in predicting CI/EAS outcomes and deciding treatment choices. However, because of the extreme genetic heterogeneity of deafness, clinical application of genetic information still entails difficulties. Target exon sequencing using massively parallel DNA sequencing is a new powerful strategy to discover rare causative genes in Mendelian disorders such as deafness. We used massive sequencing of the exons of 58 target candidate genes to analyze 8 (4 early-onset, 4 late-onset) Japanese CI/EAS patients, who did not have mutations in commonly found genes including GJB2, SLC26A4, or mitochondrial 1555A>G or 3243A>G mutations. We successfully identified four rare causative mutations in the MYO15A, TECTA, TMPRSS3, and ACTG1 genes in four patients who showed relatively good auditory performance with CI including EAS, suggesting that genetic testing may be able to predict the performance after implantation.

Downregulation of ING3 mRNA expression predicts poor prognosis in head and neck cancer

Although many clinical and pathological prognostic factors such as tumor stage and lymph‐node involvement have been described, to date no reliable or clinically applicable marker or tumor aggressiveness has been identified for head and neck cancer. In an attempt to identify such a molecular prognostic marker, we analyzed the mRNA expression status of ING3 by quantitative reverse transcription–polymerase chain reaction. We also examined p53 mutation status and investigated its relationship with ING3, as well its clinicopathological characteristics. About half of the 71 tumor samples demonstrated downregulation of ING3 compared to their matched normal counterparts. Although most clinicopathological variables were not significantly related to ING3 downregulation or p53 mutation status, a significant relationship was detected in terms of overall survival between the cases with low and normal to high ING3 expression. At 5 years follow up, approximately 60% of the patients with normal to high ING3 expression survived, whereas this was 35% in the patients with low ING3 expression. Multivariate analysis also showed downregulation of ING3 as an independent prognostic factor for poor overall survival. These results reveal that ING3 would function as a potential tumor suppressor molecule and that low levels of ING3 may indicate an aggressive nature of head and neck cancer. (Cancer Sci 2008; 99: 531–538)

Effect of single-drug treatment on idiopathic sudden sensorineural hearing loss

OBJECTIVES In order to evaluate the effect of a medical administration for the sudden deafness patients, single-drug treatment for idiopathic sudden sensorineural hearing loss (ISSHL) was assessed at multi-centers participating in the Acute Severe Hearing Loss Study Group sponsored by the Ministry of Health, Labor and Welfare of Japan. METHODS The subjects consisted of ISSHL patients who were (1) 20 years of age or older, (2) diagnosed within 2 weeks after the onset of hearing loss, (3) showing a mean hearing level of 40-90 dB at five frequencies from 250 to 4000 Hz, (4) previously untreated, and (5) with normal for age in hearing of the opposite ear. The drugs used in this study were ATP, alprostadil, hydrocortisone and amidotrizoate, which were administered intravenously, and beraprost sodium and betamethasone, which were given orally. Two drugs were assigned to each center, one of which was selected according to the code hidden in envelopes and administered for 1 week. The treatment after the single-drug administration was conducted at the discretion of each center. The hearing gain and recovery rate at 1 week after the initiation of single-drug treatment and at 1 month or over when the hearing level was fixed, were evaluated based on the criteria for hearing recovery prepared by the Acute Severe Hearing Loss Study Group. RESULTS There was no statistically significant difference in the recovery rate among drugs either at 1 week after the initiation of single-drug treatment or at the time of fixed hearing level. At the time when the hearing level was fixed, a statistically significant difference in the complete recovery rate was detected only between amidotrizoate and beraprost sodium. CONCLUSION From these results, we could not find any specific drugs recommended for ISSNHL. In evaluating the effect of the drugs, however, several problems in the clinical trial for ISSHL should be considered.

Role of prostaglandin D2 and E2 terminal synthases in chronic rhinosinusitis

Background Prostaglandin (PG)D2 and E2, two major cyclooxygenase (COX) products, are generated by PGD2 synthase (PGDS) and PGE2 synthase (PGES), respectively, and appear to mediate airway inflammation.

Long-term results of tonsillectomy as a treatment for IgA nephropathy

IgA nephropathy (IgAN) is the most common form of chronic glomerulonephritis with IgA deposits present mainly in the mesangial areas. We performed a 10-year retrospective case-control study of 71 patients with IgAN to evaluate the long-term effects and prognostic factors associated with tonsillectomy. Forty-one patients (19 males and 22 females) underwent tonsillectomy (Group A) and 30 patients (13 males and 17 females) did not (Group B). These patients were followed for more than 10 years after renal biopsy. The average age at initial renal biopsy was 29.78 years in Group A and 33.0 years in Group B. The average follow-up period was 13 years and 3 months in Group A, and 12 years and 7 months in Group B. Glomerular damage demonstrated on renal biopsy was more extensive in Group A than in Group B. Prognosis after 10 years of follow-up was compared between the two groups. The clinical remission rate was 24.4% in Group A and 13.3% in Group B, the stable renal function rate was 82.9% in Group A and 70.0% in Group B, and the renal survival rate was 95.1% in Group A and 73.3% in Group B. The renal survival rate in Group A was significantly higher than that in Group B (p <0.05). Although evaluation of renal pathology based on renal biopsy was useful in predicting the long-term effects of tonsillectomy in IgAN patients, the results of tonsillar provocation tests were not.

Language development of a multiply handicapped child after cochlear implantation

The presence of additional handicaps in hearing-impaired children makes the prediction of language ability after cochlear implantation unreliable. Only limited follow-up data on developmental improvement after implantation among multiply handicapped children is available. The present study reports the course of development (audiological and linguistic) after cochlear implantation in one subject with moderate mental retardation. Preoperatively, his language development showed 34 months delay when compared to chronological age. The difference had shortened to 23 months by 2 years post-surgery. The subjects cognitive delay had not changed upon 2-year follow-up. The cochlear implant can be credited to his improvement in language development.