Supplementary Material for: <b><i>BEND4</i></b> as a Candidate Gene for an Infection-Induced Acute Encephalopathy Characterized by a Cyst and Calcification of the Pons and Cerebellar Atrophy

Three siblings born to Turkish parents from the same village had normal brain development until acute neurological deterioration between 12 months and 8 years of age. Consequent loss of all acquired motor, social, and language functions following infections was associated with a pontine cyst, calcification, and cerebellar atrophy. Exome sequencing revealed a homozygous c.1297G&gt;A (p.Gly433Ser) alteration in <i>BEND4</i>, which was predicted to be deleterious in in silico analysis tools and segregated in multiple affected individuals in the family. <i>BEND4</i> has not been associated with any existing disease. Immunofluorescence microscopy analysis of wild-type and mutant BEND4 expressing Vero cells showed nuclear and cytoplasmic localization. Wild-type BEND4 displayed a network-like distribution, whereas mutant BEND4 showed a juxtanuclear distribution pattern. Differential proteome analysis of Vero cells expressing BEND4 revealed that mutant BEND4 expression caused selective increase in reticulocalbin-1 and endoplasmic reticulum resident protein-29. Both proteins are associated with the endoplasmic reticulum and are primarily involved in protein processing and folding pathways. Any defect or stress in protein folding creates stress on cells and may cause chronic damage. This is the first study showing that pathogenic <i>BEND4</i> variants may lead to an infection-induced acute necrotizing encephalopathy as demonstrated in characteristic neuroimaging findings.三名来自同一村庄的土耳其裔父母所生育的兄弟姐妹，在12月龄至8岁期间出现急性神经功能恶化前，大脑发育均处于正常水平。感染后出现的全部获得性运动、社交及语言功能丧失，与脑桥囊肿、钙化灶及小脑萎缩存在关联。外显子组测序结果显示，<i>BEND4</i>基因存在纯合子c.1297G>A（p.Gly433Ser）变异；该变异经计算机模拟分析工具预测为有害变异，且在该家系的多名受累个体中呈共分离特征。目前尚无任何已知疾病与<i>BEND4</i>基因相关联。对表达野生型与突变型BEND4的Vero细胞进行免疫荧光显微镜分析，结果显示BEND4蛋白定位于细胞核与细胞质中。野生型BEND4呈网状分布特征，而突变型BEND4则呈现核周分布模式。对表达BEND4的Vero细胞开展差异蛋白质组分析，结果显示突变型BEND4的表达会导致网钙蛋白-1（reticulocalbin-1）与内质网驻留蛋白-29（endoplasmic reticulum resident protein-29）出现选择性上调。这两种蛋白均定位于内质网，主要参与蛋白质加工与折叠通路。蛋白质折叠过程中出现的任何缺陷或应激反应，均会对细胞造成压力，并可能引发慢性损伤。本研究首次证实，致病性<i>BEND4</i>基因变异可引发感染诱导型急性坏死性脑病，该结论在特征性神经影像学表现中得到了验证。