| Project/Area Number |
07557248
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Embryonic/Neonatal medicine
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| Research Institution | Saitama Medical School |
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Principal Investigator |
OGAWA Yunosuke Saitama Medical School, Professor, 医学部, 教授 (90080126)
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| Co-Investigator(Kenkyū-buntansha) |
ITAKURA Yukino Saitama Medical School, Instructor, 医学部, 助手 (70223071)
KANEKO Koji Saitama Medical School, Instructor, 医学部, 助手 (30224596)
ARAKAWA Hiroshi Saitama Medical School, Instructor, 医学部, 助手 (90271238)
NAKAMURA Toshihiko Saitama Medical School, Instructor, 医学部, 助手 (30255137)
SHIMIZU Hiroshi Saitama Medical School, Lecturer, 医学部, 講師 (90260843)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | pulmonary surfactant / congenital alveolar proteinosis / surfactant proteins / surfactant protein B deficiency / polymerase chain reaction / gene analysis / serum proteins / 新生児呼吸障害 / サーファクタント蛋白質B遺伝子 / 先天性肺SP-B欠損症 / 先天性SP-B欠損症 |
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| Research Abstract |
Surfactant protein B deficiency is an inherited disease of full-term newborn infants which leads to lethal respiratory failure within the first year of life and is refractory to mechanical ventilation, surfactant therapy, glucocorticoid induction of SP-B production, and extracorporeal membrane oxygenation. The inheritance is autosomal recessive. Two mutations, l2lins2 and R236C have been identified in exons of SP-B gene to date. The l2lins2 mutation is a substitution of three bases (GAA) for the single nucleotide (C) at position 375. The net gain of two bases causes a frameshift and introduces a premature signal for termination of translation after codon 214. The R236C mutation was observed in an infant with a compound heterozygous deficiency, l2lins2 mutation in one allele and the new point mutation in the other allele, a T for C substitution in codon 236, resulting in the substitution of a cysteine for arginine normally encoded by codon 236.
We have developed a genetic diagnosis of th
… More
e mutations, l2lins2 and R236C, in congenital SP-B deficiency. A rapid procedure for microextraction of genomic DNA from whole blood was developed. Using site-directed mutagenesis by overlap extension, the l2lins2 mutation (C->GAA) was introduced into a PCR product. The R236C mutation (C->T) was also introduced using the same technique. The introduction of the mutations in SP-B genome was confirmed by the DNA sequencing after thermal cycling. The mutants were used as a positive control of the mutations. We also developed a rapid procedure for microextraction of genomic DNA from a paraffin-embedded lung tissue. We examined the indicated genomic SP-B mutations in patients with congenital alveolar proteinosis. Furthermore, hydrophilic surfactant proteins, SP-A and SP-D, in sera were determined in a patient with congenital alveolar proteinosis. Those surfactant proteins were measured using enzyme-linked immunosorbent assay with monoclonal antibodies against human SP-A and SP-D, respectively. We could detect a significant amount of SP-A and SP-D in sera, suggesting that the measurement of the surfactant proteins may provide a useful tool to evaluate the respiratory disorder. Less
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