Supplementary Material for: Molecular genetic analysis and growth hormone treatment in a three-generation Chinese family with Tricho-rhino-phalangeal syndrome I

Background: Tricho-rhino-phalangeal syndrome (TRPS) is a rare genetic disorder characterized by craniofacial and skeletal abnormalities, which is caused by variations in the TRPS1 gene. Methods: Clinical information and follow-up data were collected. Whole-exome sequencing (WES) was performed for variations and validated by Sanger sequencing. Bioinformatic analysis was performed to predict the pathogenicity of the identified variation. Moreover, wild-type and mutated TRPS1 vectors were constructed and transfected into human embryonic kidney (HEK) 293T cells. Immunofluorescence experiments were performed to assess the localization and expression of the mutated protein. Western blot analysis and RT-qPCR were used to detect the expression of downstream genes. Results: The affected family members had typical craniofacial phenotype including sparse lateral eyebrows, pear-shaped nasal tip, and large prominent ears, plus skeletal abnormalities including short stature and brachydactyly. WES and Sanger sequencing identified the TRPS1 c.880_882delAAG variation in affected family members. In vitro functional studies showed that the TRPS1 variation did not affect the cellular localization and the expression of TRPS1, but the transcriptional repression effect of the TRPS1 on the RUNX2 and STAT3 was disturbed. The proband and his brother have been treated with growth hormone (GH) for two years until now, and we observed the improvement of the linear growth in both. Conclusions: The variation of c.880_882delAAG in TRPS1 was responsible for the pathogenesis of the Chinese family with TRPS I. The treatment of GH could be beneficial for the height outcome in TRPS I patients, and earlier initiation and longer duration of the therapy in prepubertal or early pubertal stage could be associated with better height outcomes.背景：毛发-鼻-指（趾）综合征（Tricho-rhino-phalangeal syndrome, TRPS）是一种罕见的遗传性疾病，以颅面部与骨骼异常为主要特征，由TRPS1基因变异所致。 方法：本研究收集患者的临床资料与随访数据，对其实施全外显子测序（whole-exome sequencing, WES）以检测基因变异，并通过桑格测序（Sanger sequencing）对检测到的变异进行验证；采用生物信息学分析方法预测所鉴定变异的致病性。此外，构建野生型与突变型TRPS1载体，将其转染至人胚肾293T细胞（human embryonic kidney 293T, HEK293T）；通过免疫荧光实验评估突变蛋白的定位与表达水平，利用蛋白质免疫印迹（Western blot）分析与实时荧光定量PCR（RT-qPCR）检测下游基因的表达情况。 结果：受累家族成员表现出典型的颅面部表型：外侧眉毛稀疏、鼻尖呈梨形、耳大且突出，同时伴随骨骼异常：身材矮小与短指（趾）症。全外显子测序与桑格测序结果证实，受累家族成员携带TRPS1基因c.880_882delAAG变异。体外功能研究显示，该TRPS1变异不会影响TRPS1的细胞定位与蛋白表达，但可扰乱TRPS1对RUNX2及STAT3的转录抑制作用。先证者与其兄弟迄今已接受生长激素（growth hormone, GH）治疗两年，观察到二者的线性生长均得到改善。 结论：TRPS1基因c.880_882delAAG变异是该中国毛发-鼻-指（趾）综合征I型（TRPS I）家系的致病原因。生长激素治疗对TRPS I患者的身高获益具有积极作用，于青春期前或早期青春期启动治疗且疗程更长者，或可获得更优的身高结局。