CN105803078A - Cryba4基因在检测先天性ccmc中的应用 - Google Patents

Cryba4基因在检测先天性ccmc中的应用 Download PDF

Info

Publication number
CN105803078A
CN105803078A CN201610256564.0A CN201610256564A CN105803078A CN 105803078 A CN105803078 A CN 105803078A CN 201610256564 A CN201610256564 A CN 201610256564A CN 105803078 A CN105803078 A CN 105803078A
Authority
CN
China
Prior art keywords
gene
cryba4
application
ccmc
primer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201610256564.0A
Other languages
English (en)
Other versions
CN105803078B (zh
Inventor
王君怡
王殿强
陈鹏
孙大鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SPECIALTY OF OPHTHALMOLOGY RESEARCH INSTITUTE SHANDONG PROV
Original Assignee
SPECIALTY OF OPHTHALMOLOGY RESEARCH INSTITUTE SHANDONG PROV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SPECIALTY OF OPHTHALMOLOGY RESEARCH INSTITUTE SHANDONG PROV filed Critical SPECIALTY OF OPHTHALMOLOGY RESEARCH INSTITUTE SHANDONG PROV
Priority to CN201610256564.0A priority Critical patent/CN105803078B/zh
Publication of CN105803078A publication Critical patent/CN105803078A/zh
Application granted granted Critical
Publication of CN105803078B publication Critical patent/CN105803078B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明的目的是提供一种CRYBA4基因在制备检测先天性白内障‑小角膜综合征(CCMC)诊断制品中的应用，本发明提供了CRYBA4基因的新的用途，从而提供了一种有效的进行先天性CCMC疾病基因诊断、产前基因筛查及遗传咨询的途径，应用效果表明本发明所提供的基因的SNP位点及检测引物可以有效的用于临床患者及胎儿绒毛或羊水进行CRYBA4基因突变位点的快速检测。

Description

CRYBA4基因在检测先天性CCMC中的应用
技术领域
本发明属于基因诊断技术领域，具体涉及β晶状体蛋白A4(CRYBA4)基因在检测先天性白内障-小角膜综合征(CCMC)中的应用。
发明背景
先天性白内障是导致儿童低视力和致盲的常见眼病，发病率约为0.01％～0.06％，其中约50％与遗传有关，其遗传方式以常染色体显性遗传最为常见。先天性白内障可独立发生，也可作为眼部或全身综合征的伴发症状，其中12％-18％的先天性白内障患者常发生合并小角膜症状。白内障-小角膜综合征(Cataract-microcorneasyndrome,CCMC,OMIM116200)是一种先天发育异常性眼病，常累及双眼，表现为不同表型的白内障及角膜横径小于10mm，常伴发眼前节发育不良，如虹膜缺损、Peters异常、瞳孔异位及眼球震颤等，这些多发畸形常使大多数患者丧失视力，是先天性致盲的重要原因之一。并且和单纯先天性白内障相比，伴发小角膜的患者更易患青光眼，并造成不可逆转的视觉损害。目前对于CCMC治疗手段有限、致盲率很高，给全社会带来沉重的经济负担。
先天性CCMC的病因及病理机制尚不清楚，目前世界上研究较少。国内外的遗传学研究结果表明该病很大程度上是由于遗传因素导致的。CCMC具有显著的遗传异质性，存在多个候选致病基因。到目前为止对CCMC的致病基因的了解仍然有限，迄今为止已知的基因仅能解释不到30％的患者的发病。这已经成为CCMC的发病机制的研究以及针对病因的诊断和治疗研究的瓶颈。这种现状促使进一步去发现和了解该病新的致病基因，为后续的基因诊断、产前诊断及基因治疗提供基础。
发明内容
本发明的目的是提供一种CRYBA4基因在制备检测斑块状角膜营养不良诊断制品中的应用，从而弥补现有技术的不足。
申请人从一个5代呈常染色体隐性遗传的斑块状角膜营养不良家系中，对该家系全部成员进行了CRYBA4基因的测序，找到了该家系的致病基因CHST6存在遗传上的致病突变，从而促成了本发明。
本发明首先提供CRYBA4基因新的用途，是在制备用于检测斑块状角膜营养不良诊断制品中的应用；
上述的诊断制品，作为实施例的优选，是检测斑块状角膜营养不良的引物或探针。
上述的引物对，其引物信息如下：
本发明还提供一种用于检测CCMC的试剂盒，包含有上述的引物对中任一种或几种。
本发明还提供一种与CCMC疾病相关的SNP位点，为CRYBA4基因编码区域由起始密码子起第257位,其碱基为A或T；
其中用于检测上述SNP位点的引物信息如下：
CRYBA4-3L：GCTGGTCTAGAATGCAGSEQIDNO:5
CRYBA4-3R：CTCTGGAACCTTTGATTCSEQIDNO:6
本发明提供了CRYBA4基因的新的用途，从而提供了一种有效的进行先天性CCMC疾病基因诊断、产前基因筛查及遗传咨询的途径，应用效果表明本发明所提供的基因的SNP位点及检测引物可以有效的用于临床患者及胎儿绒毛或羊水进行CRYBA4基因突变位点的快速检测。
具体实施方式
本发明的申请人在一个先天性的CCMC家系中发现CRYBA4基因的突变位点从而促成了本发明。
CRYBA4基因由9909个核苷酸碱基组成，位于19q13.41，可转录成大约591bp的mRNA(NM_001886)，直接翻译形成196个氨基酸组成的蛋白质。
下面结合实施例对本发明进行详细的描述。
实施例1：从先天性CCMC家系中筛选CRYBA4基因的突变位点
1、提取外周血基因组DNA：
在符合国家相关政策规定，并在取样对象同意的基础上，抽取第一个家系成员外周静脉血2-5ml，放入EDTA抗凝管内，-80℃冻存备用；冻存的EDTA抗凝血在室温融化后，取500μL放于离心管，加入等体积TE(pH8.0)，混匀，4℃，10000rpm离心10分钟，弃上清。
加入180μLTE、20μLSDS(10％)、8μL蛋白酶K(l0mg/ml)混匀，置于37℃水浴过夜。从水浴中取出样品，瞬时离心沉淀样本。在反应管中加入等体积的Tris-饱和酚(约300μL)，充分混匀，室温下10000rpm离心10分钟，吸取上清液(约300μL)至一新离心管中。重复酚抽提一次，吸取上清液至一新离心管中。
加入等体积的Tris饱和酚:氯仿混合液(酚、氯仿各150μL)，混匀，室温10000rpm离心10分钟，转移上清液至一新离心管。
加入等体积的Tris饱和酚:氯仿:异戊醇混合液(酚、氯仿、异戊醇各100μL)，混匀，室温10000rpm离心10分钟，转移上清液至一新离心管。
加入l/10体积3mol/L、pH5.2醋酸钠(约30μL)，2倍体积预冷100％乙醇，轻轻混合，可见白色絮状沉淀。室温10000rpm离心10分钟，使DNA沉淀于管底，弃上清。
向DNA沉淀加入70％乙醇，漂洗一次，室温7000rpm离心5分钟，弃上清，置于室温中挥发剩余乙醇，最后加入50μLTE(pH8.0)，4℃过夜溶解DNA。
对提取的DNA行琼脂糖胶电泳，并应用紫外分光光度计在260nm和280nm比色，检测DNA纯度及浓度。
2.直接测序法验证该家系内患者CRYBA4基因的突变
PCR扩增目的片段：反应条件与反应体系：
(1)PCR反应条件：94℃3min；94℃40sec，55±2℃40se,72℃60sec，30-35cycles；72℃10min。
(2)反应体系：(TAKARALATaqpolymerase)
应用该反应体系分别进行每名家系成员的基因组DNA模板与该CRYBA4引物的扩增反应。
PCR产物测序：应用常规Sanger测序法对上述PCR产物进行测序，该家系中三名患者的CRYBA4基因编码区域由起始密码子起第257个碱基发生了杂合突变，由A突变为T，引起CRYBA4蛋白第86位由丝氨酸突变为苯丙氨酸，应用点突变预测程序SIFT(SortingIntolerantFromTolerant)预测发现，该突变导致了CRYBA4蛋白功能“damaging”级的损坏，从而引起了该家系中CCMC的发生。多次测序结果表明该突变位点并不是因为扩增或测序错误引进的。在200例正常当地人群及该家系中正常人的外周血基因组DNA样本中进行该位点的突变筛查,未发现该突变。
在山东省眼科研究所暨青岛眼科医院收集诊断明确的先天性CCMC散发患者一名，提取其外周血基因组DNA，应用该患者的DNA模板与CRYBA4-3L和CRYBA4-3R引物进行PCR扩增，应用常规Sanger测序法对上述PCR产物进行测序，该患者的CRYBA4基因存在本发明中发现的SNP突变，即编码区域由起始密码子起第257个碱基发生了杂合突变，由A突变为T，引起CRYBA4蛋白第86位由丝氨酸突变为苯丙氨酸。
上述结果表明CRYBA4基因可以用来检测患者是否具有潜在患CCMC的危险。通过将检测者的CRYBA4基因的各外显子片段于正常的对应片段比较，确定待检测者的患病风险。

Claims (7)

1.一种CRYBA4基因的应用，其特征在于，所述的应用是在制备用于检测先天性白内障-小角膜综合征的诊断制品中的应用。
2.如权利要求1所述的应用，其特征在于，所述的诊断制品为PCR检测引物。
3.一种用于检测先天性白内障-小角膜综合征的引物对，所述的引物对的信息如下：
4.一种用于检测先天性白内障-小角膜综合征的试剂盒，其特征在于，所述的试剂盒包含有权利要求3所述的引物对中的任一种或几种。
5.一种与先天性白内障-小角膜综合征疾病相关的SNP位点，所述的SNP位点为CRYBA4基因编码区域由起始密码子起第257位,其碱基为A或T。
6.一种检测权利要求5所述的SNP位点的方法，其特征在于，是用权利要求3所述的引物对中的任一种或几种来进行检测。
7.如权利要求6所述的方法，其特征在于，所述的引物对的信息如下：
CRYBA4-3L：GCTGGTCTAGAATGCAGSEQIDNO:5
CRYBA4-3R：CTCTGGAACCTTTGATTCSEQIDNO:6。
CN201610256564.0A 2016-04-22 2016-04-22 Cryba4基因在检测先天性ccmc中的应用 Active CN105803078B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610256564.0A CN105803078B (zh) 2016-04-22 2016-04-22 Cryba4基因在检测先天性ccmc中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610256564.0A CN105803078B (zh) 2016-04-22 2016-04-22 Cryba4基因在检测先天性ccmc中的应用

Publications (2)

Publication Number Publication Date
CN105803078A true CN105803078A (zh) 2016-07-27
CN105803078B CN105803078B (zh) 2019-08-06

Family

ID=56457458

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610256564.0A Active CN105803078B (zh) 2016-04-22 2016-04-22 Cryba4基因在检测先天性ccmc中的应用

Country Status (1)

Country Link
CN (1) CN105803078B (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399564A (zh) * 2016-11-11 2017-02-15 山东省眼科研究所 Ercc8基因在先天性白内障合并圆锥角膜检测中的应用
CN107723359A (zh) * 2017-11-01 2018-02-23 郑州大学第附属医院 一种先天性白内障致病基因及其应用、检测引物、检测试剂盒

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064827A2 (en) * 2001-02-12 2002-08-22 Aventis Pharma S.A. Nucleic acids of the human abca12 gene, vectors containing such nucleic acids, and uses thereof
CN101525619A (zh) * 2009-04-07 2009-09-09 哈尔滨医科大学 前极核性白内障致病基因及其检测方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064827A2 (en) * 2001-02-12 2002-08-22 Aventis Pharma S.A. Nucleic acids of the human abca12 gene, vectors containing such nucleic acids, and uses thereof
CN101525619A (zh) * 2009-04-07 2009-09-09 哈尔滨医科大学 前极核性白内障致病基因及其检测方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ZHOU GK ET AL.: "A missense mutation in CRYBA4 associated with congenital cataract and microcornea", 《MOLECULAR VISION》 *
王海峰等: "一个常染色体显性遗传性先天性白内障家系分析", 《黑龙江医药科学》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399564A (zh) * 2016-11-11 2017-02-15 山东省眼科研究所 Ercc8基因在先天性白内障合并圆锥角膜检测中的应用
CN107723359A (zh) * 2017-11-01 2018-02-23 郑州大学第附属医院 一种先天性白内障致病基因及其应用、检测引物、检测试剂盒
CN107723359B (zh) * 2017-11-01 2021-05-14 郑州大学第一附属医院 一种先天性白内障致病基因及其应用、检测引物、检测试剂盒

Also Published As

Publication number Publication date
CN105803078B (zh) 2019-08-06

Similar Documents

Publication Publication Date Title
Littink et al. A novel nonsense mutation in CEP290 induces exon skipping and leads to a relatively mild retinal phenotype
Hansen et al. Genetic heterogeneity in microcornea-cataract: five novel mutations in CRYAA, CRYGD, and GJA8
Bisceglia et al. VSX1 mutational analysis in a series of Italian patients affected by keratoconus: detection of a novel mutation
Yzer et al. Microarray-based mutation detection and phenotypic characterization of patients with Leber congenital amaurosis
Chandra et al. A genotype-phenotype comparison of ADAMTSL4 and FBN1 in isolated ectopia lentis
Boonstra et al. Clinical and molecular evaluation of probands and family members with familial exudative vitreoretinopathy
Maubaret et al. Autosomal dominant retinitis pigmentosa with intrafamilial variability and incomplete penetrance in two families carrying mutations in PRPF8
Hameed et al. A novel locus for Leber congenital amaurosis (LCA4) with anterior keratoconus mapping to chromosome 17p13
Davidson et al. Brittle cornea syndrome ZNF469 mutation carrier phenotype and segregation analysis of rare ZNF469 variants in familial keratoconus
Rødahl et al. Variants of anterior segment dysgenesis and cerebral involvement in a large family with a novel COL4A1 mutation
Sannan et al. Correlation of novel PAX6 gene abnormalities in aniridia and clinical presentation
Gregory-Evans et al. Non-invasive anterior segment and posterior segment optical coherence tomography and phenotypic characterization of aniridia
Jang et al. Posterior polymorphous corneal dystrophy 3 is associated with agenesis and hypoplasia of the corpus callosum
Souzeau et al. PAX6 molecular analysis and genotype–phenotype correlations in families with aniridia from Australasia and Southeast Asia
CN107723359B (zh) 一种先天性白内障致病基因及其应用、检测引物、检测试剂盒
Birtel et al. NPHP1 gene-associated nephronophthisis is associated with an occult retinopathy
Chen et al. Novel variants in familial exudative vitreoretinopathy patients with KIF11 mutations and the Genotype–Phenotype correlation
CN105803078A (zh) Cryba4基因在检测先天性ccmc中的应用
CN106282383A (zh) Krt12基因在检测圆锥角膜中的应用
CN107312859A (zh) Aqp5基因在制备检测先天性白内障制品中的应用
CN103589787B (zh) Abca3基因在制备先天性ccmc诊断制品中的应用
Chang et al. A novel splice site mutation in the PAX6 gene in a Korean family with isolated aniridia
Paavo et al. Insights into PROM1-macular disease using multimodal imaging
Xiao et al. A novel PAX6 heterozygous mutation found in a Chinese family with congenital aniridia
Abouzeid et al. A new locus for congenital cataract, microcornea, microphthalmia, and atypical iris coloboma maps to chromosome 2

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant