Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery · 2003
Gene discovery using a human vestibular schwannoma cDNA library constructed from a patient with neurofibromatosis type 2 (NF2).
Клинический случайСтатьяПоддержка исследования: вне правительства СШАПоддержка службы здравоохранения СШАНазвание и аннотация: Перевод в очереди
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Halum Stacey L, Erbe Christy B, Friedland David R, Wackym Phillip A
Аннотация
BACKGROUND: Despite a strong association of schwannomin/merlin gene mutations with vestibular schwannoma formation, the regulatory mechanisms and biologic pathways involved are still largely unknown. The hypothesis of this study is that the genesis and growth characteristics of neurofibromatosis type 2 (NF2)-associated vestibular schwannomas are determined by genetic alterations that vary in gene transcript expression; this transcript expression includes oncogenic gene products that may be identified by construction and sequencing of a cDNA library from NF2-associated vestibular schwannoma.
METHODS: Approximately 3 mL of fresh tumor was obtained during resection of a 4-cm vestibular schwannoma from a patient with NF2. Poly(A)(+) mRNA was isolated, synthesized into double-stranded cDNA, and unidirectionally inserted into Uni-Zap XR (Stratagene, La Jolla, CA) bacteriophage vectors. Bacteriophage vectors containing cDNA inserts were processed into phagemids according to Uni-Zap XR protocol, and inserted vectors were sequenced and analyzed using BLAST software (National Institutes of Health, Bethesda, MD) with GenBank, EMBL, DDBJ, and PBD databases.
RESULTS: The cDNA library contained 2.4 million primary plaques. Inserts averaged 1.8 kilobases (kb) in length, with a range of 0.8 to 3.0 kb. BLAST multidatabase comparison of the sequence data obtained from 50 randomly selected clones yielded identification of 13 sequences representing known human genes and 17 sequences representing cloned sequences with unknown function. Three clones represented sequences not previously described in vestibular schwannomas but strongly implicated in oncogenesis within other tissues.
CONCLUSIONS: These data have implications for understanding the molecular mechanisms of vestibular schwannoma tumor biology. Identified genes may provide future diagnostic/prognostic markers and therapeutic targets.
METHODS: Approximately 3 mL of fresh tumor was obtained during resection of a 4-cm vestibular schwannoma from a patient with NF2. Poly(A)(+) mRNA was isolated, synthesized into double-stranded cDNA, and unidirectionally inserted into Uni-Zap XR (Stratagene, La Jolla, CA) bacteriophage vectors. Bacteriophage vectors containing cDNA inserts were processed into phagemids according to Uni-Zap XR protocol, and inserted vectors were sequenced and analyzed using BLAST software (National Institutes of Health, Bethesda, MD) with GenBank, EMBL, DDBJ, and PBD databases.
RESULTS: The cDNA library contained 2.4 million primary plaques. Inserts averaged 1.8 kilobases (kb) in length, with a range of 0.8 to 3.0 kb. BLAST multidatabase comparison of the sequence data obtained from 50 randomly selected clones yielded identification of 13 sequences representing known human genes and 17 sequences representing cloned sequences with unknown function. Three clones represented sequences not previously described in vestibular schwannomas but strongly implicated in oncogenesis within other tissues.
CONCLUSIONS: These data have implications for understanding the molecular mechanisms of vestibular schwannoma tumor biology. Identified genes may provide future diagnostic/prognostic markers and therapeutic targets.