三個(gè)X連鎖先天性特發(fā)性眼球震顫家系的分子遺傳學(xué)研究
發(fā)布時(shí)間：2018-08-18 11:09
【摘要】：目的 分析三個(gè)X連鎖先天性特發(fā)性眼球震顫(X-linked congenital idiopathic nystagmus, X-linked CIN)家系CZ、ZB和C的臨床表型特征,研究其遺傳學(xué)病因。通過連鎖分析方法對(duì)三個(gè)X-連鎖遺傳CIN家系進(jìn)行基因定位,并對(duì)候選基因FRMD7進(jìn)行突變篩查。 方法 1、臨床研究方法 1.1眼科常規(guī)檢查 常規(guī)檢查包括詢問病史、視力、屈光狀態(tài)、眼前節(jié)裂隙燈顯微鏡檢查、直接或間接眼底鏡檢查及眼底照相等。 1.2特殊檢查 視頻眼動(dòng)圖檢查或視覺誘發(fā)電位(visual evoked potential,VEP)和視網(wǎng)膜電圖(electroretinogram, ERG)檢查。 2、分子遺傳學(xué)研究方法 2.1取家系成員外周血5-8ml,采用Roche Biochemical公司的DNA分離試劑盒提取基因組DNA。 2.2連鎖分析 選取X染色體上Xq26-q27區(qū)域內(nèi)選取具有高雜合度的微衛(wèi)星標(biāo)記物DXS1047和DXS1001。對(duì)熒光標(biāo)記的微衛(wèi)星引物進(jìn)行聚合酶鏈反應(yīng)(polymerase chain reaction, PCR),在AB13130-avant全自動(dòng)遺傳分析儀上讀取微衛(wèi)星標(biāo)記物的等位基因片斷大小,用GeneMapper3.7軟件進(jìn)行分子量?jī)?nèi)標(biāo)的檢測(cè)和擴(kuò)增片段大小分析；讀取等位基因片段大小,進(jìn)行基因分型,連鎖分析采用Mlink程序(Linkage4.0軟件包)進(jìn)行兩點(diǎn)法LOD值計(jì)算。 2.3基因序列分析 對(duì)3個(gè)家系全部患病成員進(jìn)行FRMD7基因序列分析,12個(gè)外顯子的PCR擴(kuò)增產(chǎn)物在AB13130-avant全自動(dòng)遺傳分析儀上進(jìn)行直接雙向測(cè)序。 2.4FRMD7基因的巨大缺失或重排突變篩查 家系C運(yùn)用MRC-Holland公司SALSA P269FRMD7-NYS1試劑盒進(jìn)行MLPA反應(yīng)檢測(cè)是否存在FRMD7基因的巨大缺失或重排。 結(jié)果 1.1家系CZ有4個(gè)男性患者和4個(gè)女性攜帶者。家系中沒有看到疾病在男性患者之間傳遞。通過連鎖分析,家系的致病基因與DXS1001和DXS1047緊密連鎖,θ=0時(shí)LOD值分別為1.91和1.31,基因序列分析發(fā)現(xiàn)家系FRMD7基因第7外顯子存在c.623AG的雜合性基因突變,100名正常男性對(duì)照未檢測(cè)到這種突變。 1.2家系ZB有3個(gè)男性患者,4個(gè)女性患者和4個(gè)女性攜帶者。家系中沒有看到疾病在男性患者之間傳遞。通過連鎖分析,家系的致病基因與DXS1001和DXS1047緊密連鎖,0=0時(shí),LOD值分別為2.4和1.5,基因序列分析發(fā)現(xiàn)家系FRMD7基因第9外顯子存在c.837GC的雜合性基因突變,100名正常男性對(duì)照未檢測(cè)到這種突變。 1.3家系C有12個(gè)患者,包括6個(gè)男性患者和6個(gè)女性患者。家系中沒有看到疾病在男性患者之間傳遞。通過連鎖分析,家系的致病基因與DXS1047緊密連鎖,LOD值為2.42(θ=0.1),對(duì)該區(qū)域的候選基因FRMD7直接測(cè)序未發(fā)突變。對(duì)FRMD7基因采用MLPA反應(yīng)檢測(cè),仍未檢測(cè)到FRMD7基因有缺失或者重排。 結(jié)論 1、三個(gè)家系均屬于X連鎖的先天性特發(fā)性眼球震顫家系。 2、家系CZ的致病基因?yàn)镕RMD7基因,第7外顯子存在c.623AG突變,此為中國家系里首次報(bào)道的突變體。 3、家系ZB的致病基因?yàn)镕RMD7基因,第9外顯子存在c.837GC的突變,此為中國家系里首次報(bào)道的突變體。 4、通過連鎖分析家系C致病基因定位在與DXS1047連鎖的區(qū)域,但通過對(duì)該區(qū)域的候選基因FRMD7進(jìn)行直接測(cè)序和MLPA反應(yīng)均未能檢測(cè)到突變,可能在此區(qū)域存在新的眼球震顫致病基因,也可能是目前的檢測(cè)技術(shù)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R777.46
本文編號(hào)：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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R777.46
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 聶發(fā)毅;CMYA5與精神分裂癥的關(guān)聯(lián)研究及CPF遺傳家系致病位點(diǎn)的研究[D];西北大學(xué);2014年
，本文編號(hào)：2189284
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