三个X连锁先天性特发性眼球震颤家系的分子遗传学研究
发布时间：2018-08-18 11:09
【摘要】：目的 分析三个X连锁先天性特发性眼球震颤(X-linked congenital idiopathic nystagmus, X-linked CIN)家系CZ、ZB和C的临床表型特征,研究其遗传学病因。通过连锁分析方法对三个X-连锁遗传CIN家系进行基因定位,并对候选基因FRMD7进行突变筛查。 方法 1、临床研究方法 1.1眼科常规检查 常规检查包括询问病史、视力、屈光状态、眼前节裂隙灯显微镜检查、直接或间接眼底镜检查及眼底照相等。 1.2特殊检查 视频眼动图检查或视觉诱发电位(visual evoked potential,VEP)和视网膜电图(electroretinogram, ERG)检查。 2、分子遗传学研究方法 2.1取家系成员外周血5-8ml,采用Roche Biochemical公司的DNA分离试剂盒提取基因组DNA。 2.2连锁分析 选取X染色体上Xq26-q27区域内选取具有高杂合度的微卫星标记物DXS1047和DXS1001。对荧光标记的微卫星引物进行聚合酶链反应(polymerase chain reaction, PCR),在AB13130-avant全自动遗传分析仪上读取微卫星标记物的等位基因片断大小,用GeneMapper3.7软件进行分子量内标的检测和扩增片段大小分析；读取等位基因片段大小,进行基因分型,连锁分析采用Mlink程序(Linkage4.0软件包)进行两点法LOD值计算。 2.3基因序列分析 对3个家系全部患病成员进行FRMD7基因序列分析,12个外显子的PCR扩增产物在AB13130-avant全自动遗传分析仪上进行直接双向测序。 2.4FRMD7基因的巨大缺失或重排突变筛查 家系C运用MRC-Holland公司SALSA P269FRMD7-NYS1试剂盒进行MLPA反应检测是否存在FRMD7基因的巨大缺失或重排。 结果 1.1家系CZ有4个男性患者和4个女性携带者。家系中没有看到疾病在男性患者之间传递。通过连锁分析,家系的致病基因与DXS1001和DXS1047紧密连锁,θ=0时LOD值分别为1.91和1.31,基因序列分析发现家系FRMD7基因第7外显子存在c.623AG的杂合性基因突变,100名正常男性对照未检测到这种突变。 1.2家系ZB有3个男性患者,4个女性患者和4个女性携带者。家系中没有看到疾病在男性患者之间传递。通过连锁分析,家系的致病基因与DXS1001和DXS1047紧密连锁,0=0时,LOD值分别为2.4和1.5,基因序列分析发现家系FRMD7基因第9外显子存在c.837GC的杂合性基因突变,100名正常男性对照未检测到这种突变。 1.3家系C有12个患者,包括6个男性患者和6个女性患者。家系中没有看到疾病在男性患者之间传递。通过连锁分析,家系的致病基因与DXS1047紧密连锁,LOD值为2.42(θ=0.1),对该区域的候选基因FRMD7直接测序未发突变。对FRMD7基因采用MLPA反应检测,仍未检测到FRMD7基因有缺失或者重排。 结论 1、三个家系均属于X连锁的先天性特发性眼球震颤家系。 2、家系CZ的致病基因为FRMD7基因,第7外显子存在c.623AG突变,此为中国家系里首次报道的突变体。 3、家系ZB的致病基因为FRMD7基因,第9外显子存在c.837GC的突变,此为中国家系里首次报道的突变体。 4、通过连锁分析家系C致病基因定位在与DXS1047连锁的区域,但通过对该区域的候选基因FRMD7进行直接测序和MLPA反应均未能检测到突变,可能在此区域存在新的眼球震颤致病基因,也可能是目前的检测技术仍存在不足。
[Abstract]:objective
The clinical phenotypic characteristics of CZ, ZB and C in three X-linked congenital idiopathic nystagmus (X-linked CIN) families were analyzed to study their genetic causes.
Method
1, clinical research methods.
1.1 routine eye examination
Routine examinations include medical history, visual acuity, refractive status, slit lamp microscopy, direct or indirect ophthalmoscopy, and fundus photography.
1.2 special inspection
Visual evoked potentials (VEP) and electroretinogram (ERG) were examined.
2, molecular genetic research methods.
2.1 The genomic DNA was extracted from 5-8 ml peripheral blood of family members by Roche Biochemical DNA isolation kit.
2.2 linkage analysis
High heterozygosity microsatellite markers DXS1047 and DXS1001 were selected from the Xq26-q27 region of the X chromosome. Fluorescence-labeled microsatellite primers were polymerase chain reaction (PCR), and the allele fragments of the microsatellite markers were read on AB13130-avant automatic genetic analyzer. GeneMapper 3 was used. 7 software was used to detect molecular weight internal standard and analyze amplified fragment size; read allele fragment size, genotyping, linkage analysis using Mlink program (Linkage 4.0 software package) for two-point LOD calculation.
2.3 gene sequence analysis
FRMD7 gene sequence analysis was performed in all three families. PCR products of 12 exons were sequenced directly on AB13130-avant automatic genetic analyzer.
Screening for large deletion or rearrangement mutation of 2.4FRMD7 gene
Family C used MRC-Holland SALSA P269 FRMD7-NYS1 kit for MLPA reaction to detect the presence of large deletions or rearrangements of FRMD7 gene.
Result
1.1 There were 4 male patients and 4 female carriers in the CZ family. No transmission of the disease was observed between male patients in the family. The mutation was not detected in 100 normal male controls.
There were 3 male patients, 4 female patients and 4 female carriers in the ZB family. No transmission of the disease was observed between male patients in the family. The mutation of heterozygous gene was not detected in 100 normal male controls.
1.3 Family C had 12 patients, including 6 male patients and 6 female patients. No transmission of the disease was seen between male patients. Through linkage analysis, the family pathogenic gene was tightly linked to DXS1047 with a LOD value of 2.42 (theta=0.1), and no mutation was detected in the candidate gene FRMD7. MLPA reaction was used to detect the FRMD7 gene. No deletion or rearrangement of FRMD7 gene was detected.
conclusion
1, three families belonged to X linked congenital idiopathic nystagmus family.
2. The pathogenic gene of CZ is FRMD7 gene. The mutation of c.623AG in exon 7 is the first reported mutant in Chinese family.
3. The pathogenic gene of ZB is FRMD7 gene. The mutation of c.837GC in exon 9 is the first reported mutant in Chinese family.
4. By linkage analysis, the C pathogenic gene was located in the region linked to DXS1047, but no mutation was detected by direct sequencing and MLPA reaction of the candidate gene FRMD7 in this region. It is possible that there are new pathogenic genes of nystagmus in this region, and the detection technology is still inadequate.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R777.46
本文编号：2189284
[Abstract]:objective
The clinical phenotypic characteristics of CZ, ZB and C in three X-linked congenital idiopathic nystagmus (X-linked CIN) families were analyzed to study their genetic causes.
Method
1, clinical research methods.
1.1 routine eye examination
Routine examinations include medical history, visual acuity, refractive status, slit lamp microscopy, direct or indirect ophthalmoscopy, and fundus photography.
1.2 special inspection
Visual evoked potentials (VEP) and electroretinogram (ERG) were examined.
2, molecular genetic research methods.
2.1 The genomic DNA was extracted from 5-8 ml peripheral blood of family members by Roche Biochemical DNA isolation kit.
2.2 linkage analysis
High heterozygosity microsatellite markers DXS1047 and DXS1001 were selected from the Xq26-q27 region of the X chromosome. Fluorescence-labeled microsatellite primers were polymerase chain reaction (PCR), and the allele fragments of the microsatellite markers were read on AB13130-avant automatic genetic analyzer. GeneMapper 3 was used. 7 software was used to detect molecular weight internal standard and analyze amplified fragment size; read allele fragment size, genotyping, linkage analysis using Mlink program (Linkage 4.0 software package) for two-point LOD calculation.
2.3 gene sequence analysis
FRMD7 gene sequence analysis was performed in all three families. PCR products of 12 exons were sequenced directly on AB13130-avant automatic genetic analyzer.
Screening for large deletion or rearrangement mutation of 2.4FRMD7 gene
Family C used MRC-Holland SALSA P269 FRMD7-NYS1 kit for MLPA reaction to detect the presence of large deletions or rearrangements of FRMD7 gene.
Result
1.1 There were 4 male patients and 4 female carriers in the CZ family. No transmission of the disease was observed between male patients in the family. The mutation was not detected in 100 normal male controls.
There were 3 male patients, 4 female patients and 4 female carriers in the ZB family. No transmission of the disease was observed between male patients in the family. The mutation of heterozygous gene was not detected in 100 normal male controls.
1.3 Family C had 12 patients, including 6 male patients and 6 female patients. No transmission of the disease was seen between male patients. Through linkage analysis, the family pathogenic gene was tightly linked to DXS1047 with a LOD value of 2.42 (theta=0.1), and no mutation was detected in the candidate gene FRMD7. MLPA reaction was used to detect the FRMD7 gene. No deletion or rearrangement of FRMD7 gene was detected.
conclusion
1, three families belonged to X linked congenital idiopathic nystagmus family.
2. The pathogenic gene of CZ is FRMD7 gene. The mutation of c.623AG in exon 7 is the first reported mutant in Chinese family.
3. The pathogenic gene of ZB is FRMD7 gene. The mutation of c.837GC in exon 9 is the first reported mutant in Chinese family.
4. By linkage analysis, the C pathogenic gene was located in the region linked to DXS1047, but no mutation was detected by direct sequencing and MLPA reaction of the candidate gene FRMD7 in this region. It is possible that there are new pathogenic genes of nystagmus in this region, and the detection technology is still inadequate.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R777.46
【引证文献】
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，本文编号：2189284
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