MOLECULAR PATHOPHYSIOLOGY OF GROWTH DISORDERS

我们已经对分子的作用进行了一项基于患者的研究。 患有生长障碍的个体存在缺陷。我们假设有一个子集 家族性重度分离生长激素(GH)患者的比例 缺乏症是一种影响生长激素轴的基因突变。 通过详细的内分泌评估进行仔细的表型鉴定，我们限制了 生长激素释放激素的潜在候选基因， GHRH受体(GHRHR)、生长激素促分泌剂(GHS)、GHS 受体(GHSR)和生长激素(GH1)。 我们招募和分析家族性遗传连锁的家系， 严重矮小：基因组中包含其中一个区域的严重矮小 生长激素轴的组件。然后对候选基因进行鉴定，外显子- 副外显子，最初通过单链构象分析(SSCA)和 然后通过对受影响的基因的构象唯一外显子进行直接测序 以及未受影响的家庭成员。突变状态的确认是 由检测到的限制性片段长度(RFLP)分析提供 序列变异，包括新的限制酶切位点。 这项协议需要知道一个人的遗传位置 候选基因及其优良的遗传结构。这些基因的基因组 结构未知(例如GHRHR和GHSR)将得到充分表征 由我们努力使他们能够如上所述进行筛查。 我们也在招募患有生长激素分泌肿瘤的个人 确定分子缺陷在生长激素过度/肢端肥大症中的作用。我们是 逆转录肿瘤组织总RNA以获得用于治疗肿瘤的基因 候选基因，包括GHRHR、GHSR和生长激素 因子-1(GHF-1，又称PIT-1转录因子)。在案件中 在没有RNA的情况下，我们依靠SSCA，然后进行直接测序 基因组DNA以确定导致生长激素的激活突变 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.