Surfactant protein B deficiency in neonatal respiratory disease

肺表面活性物质蛋白B缺乏症是足月新生儿的一种遗传性疾病，在出生一年内导致致死性呼吸衰竭，对机械通气、表面活性物质治疗、糖皮质激素诱导产生SP-B和体外膜氧合无效。该遗传为常染色体隐性遗传。到目前为止，已在SP-B基因外显子上发现了两个突变，L2lins2和R236C。L2lins2突变是用三个碱基(GAA)取代第375位的单核苷酸(C)。在密码子214之后，两个碱基的净增益导致了移码，并引入了提前终止翻译的信号。在1例复合性杂合性缺乏症的婴儿中发现了R236C突变，其中一个等位基因发生了L2lins2突变，另一个等位基因出现了新的点突变，即第236密码子中C的T替换，导致半胱氨酸取代了通常由密码子236编码的精氨酸。我们建立了TH…的基因诊断先天性SP-B缺乏症患者存在较多的E突变，如L2lins2和R236C。建立了一种从全血中微量提取基因组DNA的快速方法。利用重叠延伸定点突变技术，将L2lins2突变(C&GT；GAA)引入到PCR产物中。使用相同的技术也引入了R236C突变(C-&gt；T)。通过热循环后的DNA测序证实了SP-B基因突变的引入。这些突变体被用作突变的阳性对照。我们还开发了一种从石蜡包埋的肺组织中快速提取基因组DNA的程序。我们检测了先天性肺泡蛋白沉积症患者的SP-B基因突变。此外，还检测了一例先天性肺泡蛋白沉积症患者血清中的亲水性表面活性蛋白SP-A和SP-D。分别用抗人SP-A和SP-D的单抗，用酶联免疫吸附试验检测这些表面活性蛋白的含量。我们可以在血清中检测到大量的SP-A和SP-D，提示表面活性物质蛋白的测定可能为呼吸疾病的评估提供有用的工具。较少

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