A homozygous MITF mutation leads to familial Waardenburg syndrome type 4

Waardenburg syndrome (WS) is a genetic disorder characterized by hearing loss and pigmentary abnormalities with variable penetrance. Though heterozygous mutations in MITF are a major cause for Waardenburg syndrome type 2 (WS2), homozygous mutations in this gene and the associated phenotype have been rarely characterized. In this study, we identified a novel p.R223H mutation in MITF in a Chinese Han family with variable WS features. Both parents carried a heterozygous p.R223H mutation. They had normal hearing, and premature greying of the hair is their only pigmentary abnormality. In contrast, their two children both carried a homozygous p.R223H mutation and had classic WS features including profound hearing loss, heterochromia irides and marked pigmentary abnormalities in hair and skin. Interestingly, the two affected children also have persistent chronic constipation since the neonatal period, symptoms suggestive of Waardenburg syndrome type 4 (WS4). Our study revealed a likely association between homozygous mutations in MITF and WS4, which implies a dosage effect for the underlying pathogenesis mechanism.     

MicroRNA-96对人视网膜色素上皮细胞增殖与迁移的影响

目的：探讨MicroRNA-96（miR-96）对人视网膜色素上皮（RPE）细胞增殖和迁移的影响。方法：实验研究。通过RNA原位杂交检测人胚胎眼（20周）石蜡切片中RPE层miR-96的表达情况。将miR-96和阴性对照（NC）通过阳离子脂质体介导转染人眼RPE细胞,采用细胞增殖实验（MTS）、流式细胞术和Transwell实验分别检测细胞增殖、细胞周期以及细胞迁移能力。通过生物信息学及Western blot法确定miR-96作用的靶基因。应用Western blot检测miR-96对细胞增殖迁移相关信号通路蛋白（Akt、ERK）及细胞周期相关蛋白（p-Cdc2、CyclinD2、p-Rb）表达的影响。组间数据比较采用独立样本t检 验。结果：MiR-96在人眼RPE细胞中有表达。MTS结果显示,转染NC、miR-96后,人眼RPE细胞的相对增殖速率分别为100%、74%±2%,差异有统计学意义（t=42.174,P=0.002）。流式细胞术检测结果显示,转染miR-96后阻滞在G1期的RPE细胞显著多于转染NC后,且差异有统计学意义（t= -18.444,P=0.003）。Transwell实验结果显示,与转染NC相比,转染miR-96能显著抑制RPE细胞的迁移,差异具有统计学意义（t=6.754,P=0.002）。进而,明确了MITF是miR-96作用的靶基因。Western blot检测结果显示,转染miR-96后细胞中细胞周期相关蛋白p-Rb（t=11.211,P=0.002）、p-Cdc2（t=9.133, P=0.003）、CyclinD2（t=7.542,P=0.005）以及迁移相关信号通路蛋白p-ERK（t=16.699,P<0.001）,p-Akt （t=23.552,P<0.001）的表达水平均降低。结论：miR-96通过作用于靶基因MITF,并调控细胞周期和迁移相关蛋白的表达从而抑制人RPE细胞的增殖和迁移。     

Effect of natural extract eye drops in dry eye disease rats

AIM: To reveal a novel MITF gene mutation in Waardenburg syndrome (WS), which is an autosomal dominant inherited neurogenic disorder that consists of various degrees of sensorineural deafness and pigmentary abnormalities in the eyes, hair and skin. METHODS: The genetic analysis of the Chinese family was conducted by whole-exome sequencing, then the results were confirmed by Sanger sequencing. RESULTS: WS is classified into type I to IV, which are identified by the W index, clinical characteristics and additional features. The MITF gene mostly accounts for WS type II. In this study, a de novo heterozygous mutation in the MITF gene, c.638A>G in exon 7, was identified in the patient diagnosed with WS type I features, as the W index was 2.17 (over 2.10), with dystrophia canthorum, congenital bilateral profound hearing loss, bilateral heterochromia irides, premature greying of the hair, and excessive freckling on the face at birth. She also underwent refractive errors and esotropia, reduced pigmentation of the choroid and visible choroid vessels. The mutation was not found in previous studies or mutation databases. CONCLUSION: The novel mutation in the MITF gene, which altered the protein in amino acids 213 from the glutamic acid to glycine, is the genetic pathological cause for WS features in the patient. Those characteristics of this family revealed a novel genetic heterogeneity of MITF in WS, which expanded the database of MITF mutations and offered a possible in correcting the W index value of WS in distinct ethnicities. Moreover, ocular symptoms should be emphasized in all types of WS patients.     

Ⅱ型Waardenburg综合征患儿二例基因诊断分析

目的通过对2例中国云南地区Ⅱ型Waardenburg综合征(WS2)患儿相关基因突变位点的分析,探讨WS2可能的分子生物学致病原因。方法经知情同意,分别于2018年10月和2019年5月对2例WS2先证者及家系成员进行病史采集、体格检查、听力学评估及颞骨CT检查。获取外周血,提取基因组DNA, 高通量测序方法对MITF、PAX3、SOX10、SNAI2、END3、ENDRB、KITLG基因编码区的全部外显子及部分内含子和启动子进行检测,对先证者及其父母进行突变位点的Sanger测序验证分析。结果先证者1检测出MITF基因第7外显子c.641643delGAA突变,导致氨基酸改变p.214delR,为整码移码突变,患儿双亲MITF基因序列测序分析该位点无变异,此位点为自发突变。先证者2检测出MITF基因1~9号外显子大片段杂合缺失,影响蛋白质功能的正常发挥。结论基因诊断是确诊Waardenburg综合征的重要依据,MITF基因c.641643delGAA杂合突变和MITF基因1~9号外显子大片段杂合缺失可能是本研究2例WS2患儿的分子生物学致病原因。     

Waardenburg综合征Ⅱ型中国家系MITF基因突变分析

目的 探讨Waardenburg综合征Ⅱ型中国家系的临床和分子遗传学特征。方法收集两个Waardenburg综合征Ⅱ型中国家系详细的临床资料并绘制家系圈谱，签署知情同意书获取血样。聚合酶链反应扩增MITF基因编码区的全部外显子，在ABI3100自动测序仪上进行正反向测序，利用GeneTool软件及遗传学网站的信息分析数据。结果Waardenburg综合征Ⅱ型临床表现变异较大，不是所有的患者均满足Waardenburg综合征Ⅱ型的诊断标准，眼睛色素分布异常（蓝虹膜）和正常的内眦间距是临床上最常见的表型特征。MITF基因第1，7号外显子在两个隶系中分别检测到一个错义突变和一个缺失突变，50例正常对照组均未检测到这两种突变。结论本文对两个Waardenburg综合征Ⅱ型家系做出分子水平的基因诊断，其详细的临床资料有助于对该综合征的进一步认识；新的基因突变位点不仅丰富了人类基因突变数据库，而且为更好地了解MITF基因的功能提供了新的线索。