CAJ | 학술논문

目的探讨X-连锁肾上腺脑白质营养不良高风险胎儿的产前分子诊断方法 .方法提取4个高风险家系胎儿羊水细胞基因组DNA,采用亲子鉴定试验评估母体基因组DNA污染情况,用PCR扩增先证者突变的DNA片段,然后进行PCR-限制性片段长度多态性、DNA序列测定和变性高效液相色谱对胎儿羊水基因组DNA的ABCD1基因进行分析.结果 (1)家系1:用BcnI切割799 bp的PCR产物,胎儿1与其父(正常对照)酶切模式一样,PCR产物测序未发现胎儿1的ABCD1基因有与先证者相同的突变(P560L突变);(2)家系2:用MaeI切割232 bp的PCR产物,胎儿2与其父酶切模式一样,PCR产物测序未发现胎儿2的ABCD1基因有与先证者相同的突变(Q177X突变);(3)家系3:用AciI切割271 bp的PCR产物,胎儿3与先证者酶切模式一样,PCR产物测序发现胎儿3的ABCD1基因有与先证者相同的突变(R617C突变);(4)家系4:变性高效液相色谱(DHPLC)分析269 bp的PCR产物,胎儿4与其父洗脱峰的峰型相似,而与先证者的峰型不同,PCR产物测序未发现胎儿4的ABCD1基因有与先证者相同的突变(K276E突变).结合胎儿性别判断,胎儿1、2为正常纯合子,胎儿3为肾上腺脑白质营养不良半合子,胎儿4为正常半合子.结论应用产前分子诊断的方法诊断X-连锁肾上腺脑白质营养不良高风险胎儿准确且有效.
목적 탐토X-련쇄신상선뇌백질영양불량고풍험태인적산전분자진단방법 .방법 제취4개고풍험가계태인양수세포기인조DNA,채용친자감정시험평고모체기인조DNA오염정황,용PCR확증선증자돌변적DNA편단,연후진행PCR-한제성편단장도다태성、DNA서렬측정화변성고효액상색보대태인양수기인조DNA적ABCD1기인진행분석.결과 (1)가계1:용BcnI절할799 bp적PCR산물,태인1여기부(정상대조)매절모식일양,PCR산물측서미발현태인1적ABCD1기인유여선증자상동적돌변(P560L돌변);(2)가계2:용MaeI절할232 bp적PCR산물,태인2여기부매절모식일양,PCR산물측서미발현태인2적ABCD1기인유여선증자상동적돌변(Q177X돌변);(3)가계3:용AciI절할271 bp적PCR산물,태인3여선증자매절모식일양,PCR산물측서발현태인3적ABCD1기인유여선증자상동적돌변(R617C돌변);(4)가계4:변성고효액상색보(DHPLC)분석269 bp적PCR산물,태인4여기부세탈봉적봉형상사,이여선증자적봉형불동,PCR산물측서미발현태인4적ABCD1기인유여선증자상동적돌변(K276E돌변).결합태인성별판단,태인1、2위정상순합자,태인3위신상선뇌백질영양불량반합자,태인4위정상반합자.결론 응용산전분자진단적방법 진단X-련쇄신상선뇌백질영양불량고풍험태인준학차유효.
Objective To investigate methods for prenatal molecular diagnosis of fetuses at high risk for X-linked adrenoleukodystrophy(X-ALD).Methods The amniotic fluid was obtained and genomic DNA was isolated from amniotic fluid cells.Maternal contamination was evaluated by paternity test.PCRRFLP,sequencing and denaturing high performance liquid chromatography(DHPLC)were used to detect the ABCD1 gene of fetal genome.Results In the pedigree 1,the PCR product(799 bp)of the fetus 1 and her father(normal control)could be digested with BcnI. No P560L mutation,which was present in the index patient,was detected in the ABCD1 gene from the genomic DNA of the fetus 1 using direct sequencing.In the pedigree 2,the PCR product(232 bp)of the fetus 2 and her father could not be digested with MaeI and no Q177X mutation,which was present in the propositus,was detected in the ABCD1 gene from the genomic DNA of the fetus 2 using direct sequencing.In the pedigree 3,the PCR product(271 bp)was digested with AciI.the pattern of the fetus 3 and the propositus being the same,and the R617C mutation was found in the ABCD1 gene from the genomic DNA of the fetus 3 using direct sequencing.In the pedigree 4,the PCR product(269 bp)was analyzed with the DHPLC,and the pattern of elution peaks of the fetus 4 and her father was similar,but different from that of the propositus.No K276E mutation was detectable in the ABCD1 gene from the genomic DNA of the fetus 4 by using direct sequencing.Judging from the sex of the fetuses,fetuses 1 and 2 were normal homozygotes,fetus 3 was an ALD hemizygote,and fetus 4 was a normal hemizygote.Conclusion A new protocol for X-ALD prenatal molecular diagnosis is proposed,which would ensure the accuracy of prenatal diagnosis.