先天性晚发白内障FVB小鼠突变基因定位及筛选

目的以自发突变导致的先天性晚发白内障小鼠为模型,进行遗传方式鉴定和白内障相关基因定位分析。方法首先通过显微镜观察组织切片鉴定白内障病理状态,其次通过构建家系确定先天性晚发白内障在FVB小鼠中的遗传方式,其次利用多重PCR靶向测序进行100只F2代小鼠的全基因组SNP扫描定位,最后利用全外显子测序筛选出候选突变基因。结果组织切片表明自发突变引起为典型白内障性状,全基因组扫描定位显示11号染色体上的rs4228772 SNP位点与白内障表型连锁程度最高;全外显子测序结果进一步表明11号染色体上有三个基因产生了自发突变,分别是Sfi1,Obscn和Ptrh2。结论本研究使用全基因组SNP扫描连锁分析与外显子测序相结合的策略,可在已知参考基因组的物种中快速定位基因突变,结果确定了11号染色体上的三个突变基因为先天性晚发型白内障的候选基因,并以显性方式遗传。该策略亦可应用于其它遗传背景清晰的模式哺乳动物的基因功能研究。     

LP小鼠突变基因的定位及鉴定

目的以卷尾突变C57BL/6J小鼠为研究对象,通过微卫星定位以及候选基因测序分析确定突变基因位点。方法将LP小鼠与正常C57BL/6J及C3H小鼠交配,记录了后代中卷尾与正常小鼠的数目,确定卷尾突变表型的遗传模式。微卫星D1Mit113和D1Mit149对突变基因进行了精确定位,确定候选基因。PCR扩增候选基因片段直接测序并进行序列分析。用Fsp BI(Bfa I)内切酶鉴定LP小鼠杂交后代的基因型。结果 LP突变呈单基因不完全显性遗传,在不同的遗传背景中存在表型差异;Vangl2基因编码区1345bp处碱基由C→T。结论 Vangl2基因C→T的突变是一种无义突变,导致蛋白编码提前终止,是引起卷尾突变的原因;杂交LP小鼠后代中未出现纯合子小鼠,证明Vangl2基因突变纯合子致死。     

斑马鱼小头震颤突变体mise的鉴定与基因定位

目的 以自发突变导致的小头、震颤(microcephaly and seizure,mise)斑马鱼为模型,研究其遗传方式和突变基因定位。方法 通过比较正常和突变体斑马鱼的头宽和眼球面积,鉴定突变体的小头小眼性状;通过构建家系确定突变遗传方式。对极端表型群体进行基因组重测序,随后利用分离群体分组分析(bulked segregation analysis, BSA)和竞争性等位基因特异性PCR(kompetitive allele specific PCR,KASP),一代测序等方法获得突变基因定位。结果 与野生型相比,突变体mise受精后3 d(3 days post fertilization, 3 dpf)表现为小头小眼震颤表型。经基因定位及验证明确与表型相关的突变基因为terfa。结论 本研究通过全基因组重测序结合连锁分析的方法快速定位突变基因,可为斑马鱼突变体的基因定位提供参考,为神经系统发育异常的相关基因功能分析奠定基础。     

无义突变药物治疗的研究进展

随着分子生物学和遗传学研究的不断进展,遗传性疾病越来越引起人们的关注。遗传性疾病分为单基因遗传、多基因遗传和染色体异常三大种类,多是由基因突变引起,其中基因突变根据其对基因的影响可分为三种,包括同义突变(same sense mutation)、错义突变(missense mutation)和无义突变(nonsense mutation)。无义突变是指因为某个碱基     

Gata4基因H435Y突变型小鼠模型的建立及验证

目的选择野生型C57BL/6J 小鼠为研究模型,利用CRISPR/Cas9 技术建立Gata4 基因H435Y突变型小鼠。方法针对 Gata4基因的基因序列信息设计针对Gata4基因H435Y位点的单链向导RNA（single-guideRNA,sgRNA）,经活性检测后,将具 有活性的sgRNA和Cas9体外转录成RNA,通过显微注射将sgRNA、Cas9 mRNA以及含有点突变序列的donor DNA片段注射 到小鼠受精卵中,通过PCR和基因测序方法对Gata4基因突变进行检测及鉴定,培育Gata4基因突变小鼠并分析后代的突变情 况。结果顺利构建sgRNA载体并体外转录,成功sgRNA、Cas9 mRNA以及含有点突变序列的donor DNA片段注射到受精卵 中。基因测序鉴定获得4只F0代初建鼠。选取阳性F0代小鼠与野生型C57BL/6J小鼠杂交,得到F1代鼠,再相互交配获得F2 代鼠。PCR显示F2代鼠Gata4基因H435Y突变,成功建立Gata4基因H435Y突变小鼠模型并传代繁育。结论通过CRISPR/ Cas9技术可以成功建立Gata4基因H435Y突变小鼠动物模型。     

遗传性白内障与晶体蛋白基因

先天性白内障严重影响着视觉的发育。白内障的产生是大量基因通过不同机制相互作用导致突变的最终结果,大多数遗传性白内障与晶体蛋白基因的突变有关,这些晶体蛋白基因对维持晶状体的透明性和稳定性起着至关重要的作用。现对与遗传性白内障相关的晶体蛋白基因定位、晶体蛋白的组成与结构、翻译后修饰和突变蛋白的功能进行综述。     

先天性白内障一家系中两个β-晶体蛋白基因的突变筛查

目的对一中国常染色体显性先天性粉尘状白内障家系进行β-晶体蛋白基因(cryba1、crybb1)的突变筛查。方法对一中国先天性白内障家系进行研究,通过直接测序,筛查此家系中全部患者的cryba1基因、crybb1基因外显子以及临近的内含子的剪接位点。结果直接测序后发现该粉尘状白内障家系cryba1基因和crybb1基因的外显子及其临近的内含子中,均未发现任何突变。结论该表型的先天性白内障家系并非是由这两个β-晶体蛋白基因突变引起。     

疑似白内障小鼠模型培育初报

在小鼠生产中发现了两只自然突变疑似白内障的雄性小鼠，后与BALB／c雌鼠交配，它们的后代雌雄均有疑似白内障个体出现，提示这是一个常染色体显性基因。目前用传统的方法与BALB／c回交，试图把白内障基因导入BALB／c品系中，建立一个遗传性BALB／c—Cat白内障小鼠模型。     

利用CRISPR/Cas9技术构建miRNA-29b1基因敲除小鼠

目的应用CRISPR/Cas9技术构建miRNA-29b1基因敲除小鼠。方法针对miRNA-29b1基因设计一段sgRNA,sgRNA和Cas9体外转录后显微注射至C57BL/6小鼠受精卵细胞。小鼠出生后取其基因组DNA进行测序以鉴定基因型,同时取小鼠心、肝、脾、肺、肾等脏器研磨后提取总RNA,通过real-time PCR分析miRNA-29b1在这些脏器中的表达。结果设计了20 bp的miRNA-29b1sgRNA并与Cas9一起进行了体外转录,显微注射小鼠受精卵细胞后获得miRNA-29b1基因突变小鼠。测序结果表明突变小鼠有两种基因型,一种为10 bp的缺失突变;另一种为22 bp的缺失突变,同时伴有3 bp的插入突变。与野生型小鼠相比,基因突变小鼠心、肝、脾、肺、肾等组织中miRNA-29b1表达量下降明显。结论应用CRISPR/Cas9技术成功构建miRNA-29b1基因敲除小鼠。     

Molecular genetic analysis of autosomal dominant late-onset cataract in a Chinese Family

Congenital cataract is a highly heterogeneous disorder at both the genetic and the clinical-phenotypic levels.A unique cataract was observed in a 4-generation Chinese family,which was characterized by autosomal dominant inheritance and late-onset.Mutations in the 13 known genes (CRYAA,CRYAB,CRYBB1,CRYBB2,CRYGC,CRYBA1/A3,CRYGD,Connexin50,Connexin46,intrinsic membrane protein LIM2,cytoskeletal protein BFSP2,the major intrinsic protein-MIP and the heat shock factor HSF4) have previously been demonstrated to be the frequent reason for isolated congenital cataracts,but the exact molecular basis and underlying mechanisms of congenital cataract still remain unclear.This study was designed to find whether these 13 genes developed any mutation in the family members and to identify the disease-causing gene.Polymerase chain reaction (PCR) and direct DNA sequence analysis were carried out to detect the 13 genes.The results showed that no mutation causing amino acid alternations was found in these potential candidate genes among all patients in the family,and only several single-nucleotide polymorphisms (SNPs) were identified.A transitional mutation in the fourth intron of CRYBB2 and some silent mutations in the first exon of BFSP2 and CRYGD were found in the cataract family,but further study showed that these mutations could also be found in normal controls.It was concluded that some unidentified genes may underlie the occurrence of late-onset cataract in this family.A genome-wide screening will be carried out in the next study.     

GJA8基因错义突变致先天性白内障一家系遗传分析

  目的  通过使用外显子测序定位分析一先天性白内障家系中GJA8基因致病错义突变。  方法  对2020年6月在昆明医科大学第二附属医院就诊的一个先天性白内障家系全体成员进行详细的临床眼科检查及全身查体。采集先证者及6个亲属外周血并提取基因组DNA,应用全外显子测序筛查可疑致病基因,使用生物信息工具对可疑基因突变进行致病性分析,并对家系全部成员进行Sanger测序验证候选致病突变。  结果  外显子测序及生物信息学分析显示GJA8基因存在一个错义突变c.593G > A,p.R198Q,导致其第198位氨基酸残基由谷氨酰胺取代了原有的脯氨酸。氨基酸保守性分析显示该突变影响的氨基酸在物种间高度保守。在家系全部受检者中进行的Sanger测序结果表明该突变与疾病表型共分离,可以认定该突变是该突变为该家系的致病性突变,系谱分析显示该突变所致先天性白内障呈现常染色体显性遗传。  结论  位于GJA8基因的错义突变c.593G > A,p.R198Q是导致该家系出现先天性白内障的遗传病因,遗传方式为常染色体显性遗传。     

肾癌遗传学研究进展

肾癌是一类具有多种组织类型及不同遗传背景的癌症.目前至少有10种类型的肾细胞癌.其中最常见的是透明肾细胞癌,其次是乳头状肾细胞癌、嫌色肾细胞癌和集合管细胞癌.而其它的较少见.由于肾癌具有高度的遗传异质性,现在已发现了很多染色体畸变和基因的突变都可导致肾癌.而且不同类型的肾癌,其致癌基因也不同.如VHL基因突变一般引起透明肾细胞癌,而FH基因则导致乳头状肾细胞癌.近年来,新技术的发展和应用,新的肾癌相关基因的数目也在增加.如最近的外显子组序列分析就找到了一批与透明肾细胞癌有关的基因,包括PBRM1、BAP1、SET2和JARID1c等重要的肾癌相关基因.有些透明肾细胞癌同时带有两个以上基因的突变.随着新技术的出现和研究的深入,越来越多的肾癌基因及各类肾癌标记将会被发现.     

Mutations in the ENG,ACVRL1, and SMAD4 genes and clinical manifestations of hereditary haemorrhagic telangiectasia: experience from the Center for Osler’s Disease,Uppsala University Hospital

Abstract

Aim: The aim of this retrospective single-centre study was to evaluate whether mutations in the ENG, ACVRL1, and SMAD4 genes were associated with different phenotypes in hereditary haemorrhagic telangiectasia (HHT).

Methods: The case records of 21 HHT patients with verified mutations in ENG, ACVRL1, or SMAD4 genes were reviewed. The numbers of HHT diagnostic criteria fulfilled for the three genotypes were compared, as was the prevalence of complications such as iron deficiency anaemia, gastrointestinal haemorrhage, stroke, and cerebral abscess.

Results: Our results indicate that mutations in the ENG (HHT1), ACVRL1 (HHT2), and SMAD4 genes result in different HHT phenotypes. Epistaxis debuts earlier and may be more severe in HHT1 than in HHT2. The prevalence of pulmonary arteriovenous malformations (AVM) is higher in HHT type 1, whereas hepatic AVMs are more common in HHT2. One patient with mutations in both ENG and ACVRL1 genes was identified, as were two SMAD4-mutated patients suffering from the overlapping juvenile polyposis-HHT syndrome. Nearly one in five patients in our HHT population has been diagnosed with stroke or cerebral abscess, indicating a high prevalence of cerebral complications.

Conclusion: Our results showing that ENG and ACVRL1 gene mutations result in different HHT phenotypes confirm the results from other HHT centres worldwide. Cerebral complications of HHT are common, underscoring the importance of regular screening for pulmonary AVMs and early intervention against such AVMs. We have identified an HHT patient with simultaneous mutations in the ENG and ACVRL1 genes. Surprisingly, this patient has had a mild course of the disease.     

Follicle-stimulating hormone ligand and receptor mutations, and gonadal dysfunction

In contrast to the general contention, infertility can be an inherited condition. Some of the genetic causes of male and female infertility have turned out to be due to inactivating mutations in the gonadotropin and gonadotropin receptor genes. The topic of the present text is to review current knowledge on mutations affecting the function of follicle-stimulating hormone (FSH). This gonadotropin, by binding to its specific G protein-coupled cell membrane receptor (FSHR), is important for normal gonadal function. Mutations affecting gonadotropin genes are extremely rare, but recent genetic studies have revealed that the pathogenesis of subfertility or infertility can be due to mutations in the FSH receptor (FSHR) gene. While mutations affecting FSHR are sporadic, polymorphism of the FSHR gene seems to be a common phenomenon. To date, six inactivating and only one activating mutation have been detected in the FSHR gene. In contrast to LHR gene, the majority of these mutations affect the extracellular domain of the receptor. Together with animal models using the transgenic and knock-out approaches, systematic analysis of alterations in the FSHR gene increases our knowledge on the structure and function of the FSHR and demonstrates that the integrity of each FSHR segment is required for proper expression of the fully active protein and for normal gonadal function. Mutations in the FSHR gene have different consequences in the reproductive function depending on the sex of the patient: while normal ovarian function is critically dependent on FSH, male fertility is possible with minimal or absent FSH action.     

先天性遗传性白内障与致病膜蛋白基因

白内障的产生是大量基因通过不同机制相互作用导致突变的最终结果,大多数遗传性白内障与基因亚群有关,这些编码蛋白的基因对维持晶状体的透明性和稳定性起着至关重要的作用。连接晶状体上皮和成熟晶状体纤维细胞的缝隙连接及通道在阻止结构蛋白沉积和白内障形成中起着重要作用。     

Screening of pathogenic genes in Chinese patients with arrhythmogenic right ventricular cardiomyopathy

Background Arrhythmogenic right ventricular cardiomyopathy （ARVC） is a heritable cardiac disease predominantly caused by mutations in desmosomal protein genes. Previous genetic analyses of the Chinese ARVC population are limited to small size and restriction to a single gene. This study was aimed to investigate the genotype in a large series of Chinese patients with ARVC through comprehensively screening nine ARVC-causing genes. Methods A total of 100 unrelated ARVC patients and 300 age, gender and ethnicity matched healthy controls were genetically tested with multiplexing targeted resequencing for nine previously reported ARVC-causing genes, including plakophilin-2, desmoplakin, desmoglein-2, desmocollin-2, plakoglobin, transforming growth factor beta-3, transmembrane protein 43, desmin and Lamin A/C. Results Fifty-nine mutations were identified in 64% of the patients, among which, 93% were located in desmosomal protein genes. Plakophilin-2 mutations accounted for 54% of the total and 58% of the desmosomal mutations, with a truncating mutation type making up about 2/3 of the plakophilin-2 mutations. Only four mutations were found in nondesmosomal genes; two in transmembrane protein 43 and two in transforming growth factor beta-3. Two of them （one of each gene） appeared as single missense mutations. No mutation was identified in desmin or Lamin A/C. Multiple mutations were found in 23% of the patients, with plakophilin-2 being found in 57% of the multi-mutation carriers. Conclusions Plakophilin-2 was the most common gene mutation that was identified in Chinese ARVC patients. Nondesmosomal genes should be added to desmosomal protein genes when performing molecular genetic screening in patients with suspected ARVC.     

KCNQ1和KCNH2基因与家族性猝死的关系

目的：分析离子通道基因KCNQ1和KCNH2与家族性猝死（familial sudden death,FSD)的关系，以探讨其分子遗传机制。方法：在1个FSD大家系中，利用PCR直接测序技术，对KCNQ1和KCNH2基因的所有外显子和附近的部分内含子进行序列测定。结果：KCNQ1基因存在4种突变，其中3个位于外显子区域，但均为同义突变，另外1个位于内含子区域；而KCNH2基因未发现突变。结论：在该FSD家系中，KCNQ1基因存在4种突变，但均为非致病突变，KCNH2基因无突变发生；KCNQ1和KCNH2基因以外的基因可能才是FSD致病基因。     

Genes Causing Male Infertility in Humans

There are an accumulating number of identified gene mutations that cause infertility in humans. Most of the known gene mutations impair normal puberty and subsequently cause infertility by either hypothalamic /pituitary deficiency of important tropic factors to the gonad or by gonadal genes.