Berg M A, Guevara-Aguirre J, Rosenbloom A L, Rosenfeld R G, Francke U
Department of Genetics, Stanford University School of Medicine, California.
Hum Mutat. 1992;1(1):24-32. doi: 10.1002/humu.1380010105.
Laron syndrome is an autosomal recessive condition characterized by resistance to growth hormone. We sought to determine the molecular basis of this condition in an Ecuadorean population with a high incidence of affected individuals. Growth hormone receptor gene sequences from an obligate heterozygote were amplified by the polymerase chain reaction and screened for mutations using denaturing gradient gel electrophoresis. Only exon 6 revealed homo- and heteroduplexes on denaturing gradient gels. Sequencing revealed a substitution of guanine for adenine in the third position of codon 180 that did not change the amino acid encoded. Sequencing of the exon 6-exon 7 splice junction from RNA-polymerase chain reaction amplified cellular RNA of an affected individual revealed that the substitution activates a 5' splice site 24 nucleotides upstream from the normal exon 6-intron 6 boundary. Splicing in two probands' lymphoblasts occurred virtually exclusively at the abnormal 5' splice site created by the codon 180 substitution. Exon 6 sequences from 38 patients and 47 relatives were amplified and analyzed by sequencing or dot-blot hybridization with allele-specific oligonucleotides. The substitution was detected in 74 of 76 Laron syndrome patients' GH-receptor alleles. All 26 parents and 12 of 21 unaffected siblings were heterozygous for this mutation. It was absent in 61 unrelated unaffected control individuals. We conclude that the codon 180 nucleotide substitution probably causes Laron syndrome as translation of the observed, abnormally spliced growth hormone receptor transcript would lead to the synthesis of a receptor protein with an 8 amino acid deletion from the extracellular domain.
拉伦综合征是一种常染色体隐性疾病，其特征为对生长激素有抵抗性。我们试图确定在厄瓜多尔一个患病人数高发人群中该疾病的分子基础。通过聚合酶链反应扩增一名必然杂合子的生长激素受体基因序列，并使用变性梯度凝胶电泳筛选突变。在变性梯度凝胶上，只有外显子6显示出同源双链和异源双链。测序显示，密码子180第三位的腺嘌呤被鸟嘌呤取代，这并未改变所编码的氨基酸。对一名患病个体的RNA聚合酶链反应扩增的细胞RNA中外显子6 - 外显子7剪接位点进行测序，结果显示该取代激活了正常外显子6 - 内含子6边界上游24个核苷酸处的一个5'剪接位点。两名先证者淋巴细胞中的剪接实际上仅发生在由密码子180取代产生的异常5'剪接位点。对38名患者和47名亲属的外显子6序列进行扩增，并通过测序或与等位基因特异性寡核苷酸的点杂交进行分析。在76个拉伦综合征患者的生长激素受体等位基因中，有74个检测到该取代。所有26名父母以及21名未患病同胞中的12名均为该突变的杂合子。在61名无关的未患病对照个体中未检测到该突变。我们得出结论，密码子180核苷酸取代可能导致拉伦综合征，因为观察到的异常剪接的生长激素受体转录本的翻译会导致合成一种细胞外结构域缺失8个氨基酸的受体蛋白。
