Rare genetic disorders causing specific congenital developmental abnormalities often manifest in single families. Investigation of disease-causing molecular features are most times lacking, although these investigations may open novel therapeutic options for patients. In this study, we aimed to identify the genetic cause in an Iranian patient with severe skeletal dysplasia and to model its molecular function in zebrafish embryos. The proband displays short stature and multiple skeletal abnormalities, including mesomelic dysplasia of the arms with complete humero-radio-ulna synostosis, arched clavicles, pelvic dysplasia, short and thin fibulae, proportionally short vertebrae, hyperlordosis and mild kyphosis. Exome sequencing of the patient revealed a novel homozygous c.374G > T, p.(Arg125Leu) missense variant in MSGN1 (NM_001105569). MSGN1, a basic-Helix–Loop–Helix transcription factor, plays a crucial role in formation of presomitic mesoderm progenitor cells/mesodermal stem cells during early developmental processes in vertebrates. Initial in vitro experiments show protein stability and correct intracellular localization of the novel variant in the nucleus and imply retained transcription factor function. To test the pathogenicity of the detected variant, we overexpressed wild-type and mutant msgn1 mRNA in zebrafish embryos and analyzed tbxta (T/brachyury/ntl). Overexpression of wild-type or mutant msgn1 mRNA significantly reduces tbxta expression in the tailbud compared to control embryos. Mutant msgn1 mRNA injected embryos depict a more severe effect, implying a gain-of-function mechanism. In vivo analysis on embryonic development was performed by clonal msgn1 overexpression in zebrafish embryos further demonstrated altered cell compartments in the presomitic mesoderm, notochord and pectoral fin buds. Detection of ectopic tbx6 and bmp2 expression in these embryos hint to affected downstream signals due to Msgn1 gain-of-function. In contrast to loss-of-function effects described in animal knockdown models, gain-of-function of MSGN1 explains the only mildly affected axial skeleton of the proband and rather normal vertebrae. In this context we observed notochord bending and potentially disruption of pectoral fin buds/upper extremity after overexpression of msgn1 in zebrafish embryos. The latter might result from Msgn1 function on mesenchymal stem cells or on chondrogenesis in these regions. In addition, we detected ectopic tbx6 and bmp2a expression after gain of Msgn1 function in zebrafish, which are interconnected to short stature, congenital scoliosis, limb shortening and prominent skeletal malformations in patients. Our findings highlight a rare, so far undescribed skeletal dysplasia syndrome associated with a gain-of-function mutation in MSGN1 and hint to its molecular downstream effectors.

中文翻译：

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斑马鱼作为模型研究 MSGN1 中与新型骨骼发育不良综合征相关的双等位基因功能获得变异

导致特定先天性发育异常的罕见遗传性疾病通常出现在单亲家庭中。尽管这些研究可能为患者提供新的治疗选择，但大多数情况下缺乏对致病分子特征的研究。在这项研究中，我们的目的是确定一名患有严重骨骼发育不良的伊朗患者的遗传原因，并对其在斑马鱼胚胎中的分子功能进行建模。先证者表现出身材矮小和多种骨骼异常，包括手臂中段发育不良，伴有肱骨-桡骨-尺骨完全联结、锁骨弓形、骨盆发育不良、腓骨短而薄、椎骨比例短、脊柱前凸过度和轻度后凸。该患者的外显子组测序显示 MSGN1 (NM_001105569) 中存在新的纯合 c.374G > T, p.(Arg125Leu) 错义变异。MSGN1 是一种碱性螺旋-环-螺旋转录因子，在脊椎动物早期发育过程中，在体前中胚层祖细胞/中胚层干细胞的形成中发挥着至关重要的作用。最初的体外实验显示了新变体在细胞核中的蛋白质稳定性和正确的细胞内定位，这意味着保留了转录因子功能。为了测试检测到的变异的致病性，我们在斑马鱼胚胎中过表达野生型和突变型 msgn1 mRNA 并分析 tbxta (T/brachyury/ntl)。与对照胚胎相比，野生型或突变型 msgn1 mRNA 的过表达显着降低尾芽中 tbxta 的表达。注射突变型 msgn1 mRNA 的胚胎表现出更严重的影响，这意味着功能获得机制。通过在斑马鱼胚胎中克隆 msgn1 过度表达对胚胎发育进行体内分析，进一步证明了前体中胚层、脊索和胸鳍芽中细胞区室的改变。在这些胚胎中检测到异位 tbx6 和 bmp2 表达表明下游信号因 Msgn1 功能获得而受到影响。与动物击倒模型中描述的功能丧失效应相反，MSGN1 的功能获得解释了先证者的唯一轻度受影响的轴向骨骼和相当正常的椎骨。在这种情况下，我们观察到斑马鱼胚胎中 msgn1 过度表达后脊索弯曲和胸鳍芽/上肢的潜在破坏。后者可能是由 Msgn1 对间充质干细胞或这些区域的软骨形成的功能造成的。此外，我们在斑马鱼中获得 Msgn1 功能后检测到异位 tbx6 和 bmp2a 表达，这与患者身材矮小、先天性脊柱侧凸、肢体缩短和显着骨骼畸形有关。我们的研究结果强调了一种罕见的、迄今为止尚未描述的骨骼发育不良综合征，该综合征与 MSGN1 的功能获得性突变相关，并暗示了其分子下游效应器。