MOLECULAR PATHOPHYSIOLOGY OF GROWTH DISORDERS

我们进行了一项以患者为基础的研究， 生长障碍患者的缺陷。我们假设一个子集 家族性严重孤立性生长激素（GH） 缺乏有一个基因突变损害生长激素轴。 通过详细的内分泌评估进行仔细的表型分析，我们限制了 生长激素释放激素（GHRH）的潜在候选基因， GHRH受体（GHRHR），生长激素促分泌素（GHS），GHS 受体（GHSR）和生长激素（GH 1）。 我们招募并分析家庭的遗传连锁， 严重的身材矮小的基因组区域包含一个 GH轴的分量。然后对候选基因进行表征，外显子- 按外显子，最初通过单链构象分析（SSCA）， 然后通过直接测序受影响的基因中构象独特的外显子， 和未受影响的家庭成员。突变状态的确认是 通过限制性片段长度（RFLP）分析提供， 序列变体，包括新的限制性位点。 该方案需要了解一个基因的遗传位置， 候选基因及其精细遗传结构。那些基因组 结构未知（例如GHRHR和GHSR）将得到充分表征 我们努力使他们的筛选如上所述。 我们还招募患有生长激素分泌肿瘤的个体， 确定GH过量/肢端肥大症中分子缺陷的作用。我们 逆转录来自肿瘤组织的总RNA以获得用于 候选基因，包括GHRHR、GHSR和生长激素 因子-1（GHF-1，也称为PIT-1转录因子）。情况下 在RNA不可用的情况下，我们依靠SSCA，然后直接测序 的基因组DNA，以确定激活突变，导致生长激素 过度/肢端肥大症。

We have undertaken a patient-based study of the role of molecular defects in individuals with growth disorders. We hypothesize that a subset of individuals with familial severe isolated Growth Hormone (GH) deficiency have a mutation in a gene compromising the Growth Hormone axis. Careful phenotyping via a detailed endocrine evaluation, we limit the potential candidate genes to the Growth Hormone Releasing Hormone (GHRH), the GHRH Receptor (GHRHR), the Growth Hormone Secretagogue (GHS), the GHS receptor (GHSR) and Growth Hormone (GH1). We recruit and analyze families for genetic linkage of familial, severe short stature to a region of the genome containing one of the components of the GH axis. The candidate gene is then characterized, exon- by-exon, initially by Single Strand Conformational Analysis (SSCA) and then by direct sequencing of the conformationally unique exon in affected and unaffected members of a family. Confirmation of mutational status is furnished by restriction fragment length (RFLP) analysis which detects sequence variants, including novel restriction sites. This protocol requires knowledge of the genetic position of a candidate gene, and its fine genetic structure. Those genes whose genomic structure is not known (e.g. GHRHR and GHSR) will be fully characterized by us in an effort to enable their screening as above. We are also recruiting individuals with GH-secreting tumors to determine the role of molecular defects in GH excess/acromegaly. We are revere transcribing the total RNA from tumor tissue to obtain cDNA for candidate genes, including the GHRHR, the GHSR and the Growth Hormone Factor-1 (GHF-1, also called the PIT-1 transcription factor). In cases where RNA is not available, we rely on SSCA followed by direct sequencing of genomic DNA to identify activating mutations resulting in GH excess/acromegaly.