KOBAYASHI Miyuki The University of Tokyo, Faculty of Medicine, Lecturer, 医学部附属病院, 講師 (60205391)
IDA Kohmei The University or Tokyo, Faculty of Medicine, Assistant, 医学部附属病院, 助手 (60313128)
HAYASHI Yasuhide The University of Tokyo, Faculty of Medicine, Lecturer, 医学部附属病院, 講師 (30238133)
滝田 順子 東京大学, 医学部附属病院, 助手
|Budget Amount *help
¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
To identify candidate tumor suppressor genes on chromosome 1p36 for neuroblastoma (NB), we screened homozygous deletions on 1p35-36 in NB cell lines by PCR according to a high-density sequence tagged site (STS)-content map spanning, approximately 35 Mb on 1p35-36. Among 25 NB cell lines, one cell line, NB-1, showed 〜 480 kb homozygously deleted region at 1p36.2. We identified 7 known genes, including the DFF45, KIF1B-β and KIF1B-α genes in this deleted region. To assess whether the inactivation of DFF45, KIF1B-β and/or KIF1B-α plays a role in the pathogenesis of NB, we examined the involvement of both these genes in NB. Six novel variant transcripts of the DFF45 gene were found in NB cell lines, but not in normal adrenal grand and peripheral blood. The discrepancy between normal transcript and those variants may affect apoptosis pathway. However, no mutations of the DFF45 gene were detected by PCR-SSCP We determined the genomic structure of the human KIF1B-β and KIF1B-α genes and found
that these genes have at least 47 exons and 23 exons, respectively. Allelic imbalance (AI) analysis of the KIF1B-β gene revealed relatively high incidence of AI (38%) in NBs. Obvious expression in all NB cell lines was detected except NB-1. Two missense mutation, 5 silent mutations and several polymorphisms of the KIF1B-β and KIF1B-α genes were observed. Although the missense mutation was located in the cargo domain, it is not known whether this mutation is functionally significant or not. Thus, these findings suggest that the DFF45, KIF1B-β and KIF1B-α genes may not be candidate tumor suppressor genes for NB.
Charcot-Marie-Tooth disease type 2A (CMT2A) was previously mapped to an interval containing KIF1B. We show that CMT2A patients contain a loss-of-function mutation in the motor domain of the KIF1B-β gene. This is clear indication that defects in axonal transport due to a mutated motor protein can underlie human peripheral neuropathies the most common inherited peripheral neuropathy.
To investigate the various genetic characteristics between the early-and advanced-stages of NB and identify the candidate genes involved in NB progression, we performed DNA microarray analysis in a total of 20 primary tumors. A two-way clustering analysis based on the expression pattern of approximately 500 of 1700 genes revealed genetic subgroups in these samples. Although 9 of 13 early-stage tumors (69%) and 4 of 6 advanced-stage tumors (67%) were classified as the same cluster, respectively, the remaining tumors showed different expression profiles. This indicates that both the early-and advanced-stage tumors were heterogeneous. Based on the microarray data, we identified the API2, p19INK4D, and BAF60c genes that are predominantly expressed in either the early-or advanced-stage of NB. For better assess the prognostic value of these gene expressions in NB, real-time PCR was carried out in 50 primary tumors. The expression of both the API2 and p19INK4D genes was significantly higher in the early-stage group than the advanced-stage group (P=0.002 and 0.003, respectively), whereas the expression of the BAF60c gene was significantly reduced in the early stage group (P=0.02). Therefore, it is possible that the API2, p19INK4D, and BAF60c genes are candidates as novel prognostic markers for NB. Less