Genetic and functional studies of the microphthalmia with linear skin defects (MLS) syndrome

MLS（小眼伴线状皮肤缺损）综合征是一种罕见的x染色体神经皮肤发育障碍，通常在女性中表现出来，并与男性在子宫内的死亡率有关。除了面部和颈部的线状皮肤缺陷外，小眼症和硬角膜还可出现其他临床特征，如先天性膈疝（CGH）。已知存在杂合突变的疾病基因HCCS、COX7B和NDUFB11编码线粒体呼吸链蛋白。缺乏HCCS、COX7B或NDUFB11的细胞表现出呼吸链酶的组装和活性缺陷，并增加细胞死亡。在Medaka中，hccs的下调通过由活性氧（ROS）触发的凋亡无关的caspase-9激活导致细胞死亡增加。在第二个资助期，我们将重点研究工作包，以进一步阐明MLS综合征的遗传基础和病理机制。通过对CGH患者中HCCS基因的测序和拷贝数分析，我们将回答HCCS突变是否会导致分离形式的CGH的问题。为了测试检测到的HCCS变异是否会影响呼吸链活性，我们将使用酵母互补试验。通过对6例MLS综合征患者的全外显子组测序（WES），我们旨在鉴定新的疾病基因。我们将通过功能研究确定单个序列变异的致病性或描述同一基因中不同变异的后果。在一例具有MLS综合征典型特征的男性患者中，我们发现了编码组蛋白去乙酰化酶6的x染色体基因HDAC6中的p.（A217V）变异。我们将使用患者和对照成纤维细胞生化分析组蛋白去乙酰化酶6活性和蛋白质-蛋白质相互作用。HDAC6变异对细胞死亡和呼吸链活性的影响将通过膜联蛋白V染色、ROS水平测量、线粒体膜电位分析、线粒体细胞色素c释放和半胱天冬酶活性测量来研究。类似的实验将在稳定的、缺乏ndufb11的HeLa细胞中进行。我们将通过morpholino诱导的敲低和使用CRISPR-Cas9技术来检测和表征鱼类的发育缺陷，从而生成cox7b和ndufb11的斑马鱼模型。斑马鱼突变体也将被用于研究遗传缺陷对细胞死亡、细胞增殖和氧化磷酸化的影响。

MLS (microphthalmia with linear skin defects) syndrome is a rare, X-chromosomal neurocutaneous developmental disorder that usually manifests in females and is associated with male lethality in utero. Besides linear skin defects on face and neck, microphthalmia and sclerocornea other clinical features, such as congenital diaphragmatic hernia (CGH), can be present in affected individuals. The known disease genes HCCS, COX7B and NDUFB11, in which heterozygous mutations have been identified, code for proteins of the mitochondrial respiratory chain. Cells deficient of HCCS, COX7B or NDUFB11 show defects in assembly and activity of the respiratory chain enzymes as well as enhanced cell death. Downregulation of hccs in Medaka causes increased cell death via an apoptosome-independent caspase-9 activation which is triggered by reactive oxygen species (ROS).In the second funding period, we will focus on work packages to elucidate further the genetic basis and pathomechanism underlying MLS syndrome. By sequencing and copy number analysis of the HCCS gene in patients with CGH, we will answer the question if HCCS mutations cause an isolated form of CGH. To test if detected HCCS variants affect respiratory chain activity, we will use a yeast complementation assay. By whole-exome sequencing (WES) in six patients affected by MLS syndrome, we aim to identify novel disease genes. We will determine pathogenicity of a single sequence variant or characterize consequences of different variants in the same gene by performing functional studies. By WES in a male patient with typical features of MLS syndrome, we identified the p.(A217V) variant in the X-chromosomal gene HDAC6 which encodes the histone deacetylase 6. We will use patient and control fibroblasts to biochemically analyse histone deacetylase 6 activity and protein-protein interactions. The effect of the HDAC6 variant on cell death and respiratory chain activity will be studied using annexin V staining, measurement of ROS levels, analysis of the mitochondrial membrane potential, cytochrome c release from mitochondria, and measurement of caspase activity. Similar experiments will be performed with stable, NDUFB11-deficient HeLa cells. We will generate zebrafish models for cox7b and ndufb11 by morpholino-induced knockdown and by using the CRISPR-Cas9 technology to detect and characterize developmental defects in the fishes. Zebrafish mutants will also be used to study the effect of the genetic defect on cell death, cell proliferation and oxidative phosphorylation.