中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2011年
3期
241-246
,共6页
赵燕%孟亚仙%郭奕斌%杜敏联%艾阳
趙燕%孟亞仙%郭奕斌%杜敏聯%艾暘
조연%맹아선%곽혁빈%두민련%애양
黏多糖贮积症ⅣA型%GALNS基因%新突变%鉴定
黏多糖貯積癥ⅣA型%GALNS基因%新突變%鑒定
점다당저적증ⅣA형%GALNS기인%신돌변%감정
mucopolysaccharidosis type ⅣA%GALNS gene%novel mutation%identification
目的 研究黏多糖贮积症ⅣA型(mucopolysaccharidosis type ⅣA,MPS ⅣA)患者发病的分子遗传学机制,揭示其基因型与表现型的相互关系,为产前基因诊断等创造必要的前提条件.方法 采用尿糖胺聚糖(glycosaminoglycans,GAGs)定性检测法对疑似MPS ⅣA型的先证者进行初诊,然后采用PCR及扩增产物直接测序法对先证者及其家庭成员进行突变检测.在检出GALNS基因c.1567T＞G新突变后,先后建立XspⅠ酶切鉴定法和扩增阻碍突变系统(amplification refractory mutation system,ARMS)快速特异鉴定法,对随机采集的110名正常对照与先证者及其家庭成员的GALNS基因第14外显子进行序列分析,同时采用生物信息学方法对蛋白质二级、三级结构进行预测,以及直接测定患儿GALNS酶活性的方法,对该新突变进行致病性鉴定.结果 先证者尿检呈弱阳性GAGs(±),其GALNS基因第14外显子内存在杂合的c.1567T＞G终止密码突变,第4外显子存在杂合的c.374C＞T错义突变,为两种突变的复合杂合子.其妹突变类型与先证者完全相同,其母仅在第14外显子存在杂合的终止密码突变,为该病的携带者,其父仅在第4外显子存在杂合的错义突变,也为杂合子;第14外显子的PCR产物经XspⅠ酶切后,正常对照组切出28 bp、120 bp、399 bp 3条带,而患者和携带者的母亲均切出28 bp、120 bp、148 bp和399 bp 4条带;用ARMS特异引物扩增后,正常对照组均扩增阴性,而患者及携带者均扩增阳性;蛋白质二级、三级结构预测结果显示:c.1567T＞G变异导致终止密码(TAG)突变为谷氨酸(GAG),使多肽链延长了92个氨基酸残基,导致蛋白质二、三级结构发生明显改变,而正常对照无此变化.酶活性测定的结果显示:患者的GALNS酶活性仅为8.3 nmol/17 h/mg pr,明显低于正常值(正常参考值为41.9～92.1 nmol/17h/mg pr).结论 c.1567T＞G变异是一种新的致病性突变,是引起该家系患儿发病的根本原因.
目的 研究黏多糖貯積癥ⅣA型(mucopolysaccharidosis type ⅣA,MPS ⅣA)患者髮病的分子遺傳學機製,揭示其基因型與錶現型的相互關繫,為產前基因診斷等創造必要的前提條件.方法 採用尿糖胺聚糖(glycosaminoglycans,GAGs)定性檢測法對疑似MPS ⅣA型的先證者進行初診,然後採用PCR及擴增產物直接測序法對先證者及其傢庭成員進行突變檢測.在檢齣GALNS基因c.1567T＞G新突變後,先後建立XspⅠ酶切鑒定法和擴增阻礙突變繫統(amplification refractory mutation system,ARMS)快速特異鑒定法,對隨機採集的110名正常對照與先證者及其傢庭成員的GALNS基因第14外顯子進行序列分析,同時採用生物信息學方法對蛋白質二級、三級結構進行預測,以及直接測定患兒GALNS酶活性的方法,對該新突變進行緻病性鑒定.結果 先證者尿檢呈弱暘性GAGs(±),其GALNS基因第14外顯子內存在雜閤的c.1567T＞G終止密碼突變,第4外顯子存在雜閤的c.374C＞T錯義突變,為兩種突變的複閤雜閤子.其妹突變類型與先證者完全相同,其母僅在第14外顯子存在雜閤的終止密碼突變,為該病的攜帶者,其父僅在第4外顯子存在雜閤的錯義突變,也為雜閤子;第14外顯子的PCR產物經XspⅠ酶切後,正常對照組切齣28 bp、120 bp、399 bp 3條帶,而患者和攜帶者的母親均切齣28 bp、120 bp、148 bp和399 bp 4條帶;用ARMS特異引物擴增後,正常對照組均擴增陰性,而患者及攜帶者均擴增暘性;蛋白質二級、三級結構預測結果顯示:c.1567T＞G變異導緻終止密碼(TAG)突變為穀氨痠(GAG),使多肽鏈延長瞭92箇氨基痠殘基,導緻蛋白質二、三級結構髮生明顯改變,而正常對照無此變化.酶活性測定的結果顯示:患者的GALNS酶活性僅為8.3 nmol/17 h/mg pr,明顯低于正常值(正常參攷值為41.9～92.1 nmol/17h/mg pr).結論 c.1567T＞G變異是一種新的緻病性突變,是引起該傢繫患兒髮病的根本原因.
목적 연구점다당저적증ⅣA형(mucopolysaccharidosis type ⅣA,MPS ⅣA)환자발병적분자유전학궤제,게시기기인형여표현형적상호관계,위산전기인진단등창조필요적전제조건.방법 채용뇨당알취당(glycosaminoglycans,GAGs)정성검측법대의사MPS ⅣA형적선증자진행초진,연후채용PCR급확증산물직접측서법대선증자급기가정성원진행돌변검측.재검출GALNS기인c.1567T＞G신돌변후,선후건립XspⅠ매절감정법화확증조애돌변계통(amplification refractory mutation system,ARMS)쾌속특이감정법,대수궤채집적110명정상대조여선증자급기가정성원적GALNS기인제14외현자진행서렬분석,동시채용생물신식학방법대단백질이급、삼급결구진행예측,이급직접측정환인GALNS매활성적방법,대해신돌변진행치병성감정.결과 선증자뇨검정약양성GAGs(±),기GALNS기인제14외현자내존재잡합적c.1567T＞G종지밀마돌변,제4외현자존재잡합적c.374C＞T착의돌변,위량충돌변적복합잡합자.기매돌변류형여선증자완전상동,기모부재제14외현자존재잡합적종지밀마돌변,위해병적휴대자,기부부재제4외현자존재잡합적착의돌변,야위잡합자;제14외현자적PCR산물경XspⅠ매절후,정상대조조절출28 bp、120 bp、399 bp 3조대,이환자화휴대자적모친균절출28 bp、120 bp、148 bp화399 bp 4조대;용ARMS특이인물확증후,정상대조조균확증음성,이환자급휴대자균확증양성;단백질이급、삼급결구예측결과현시:c.1567T＞G변이도치종지밀마(TAG)돌변위곡안산(GAG),사다태련연장료92개안기산잔기,도치단백질이、삼급결구발생명현개변,이정상대조무차변화.매활성측정적결과현시:환자적GALNS매활성부위8.3 nmol/17 h/mg pr,명현저우정상치(정상삼고치위41.9～92.1 nmol/17h/mg pr).결론 c.1567T＞G변이시일충신적치병성돌변,시인기해가계환인발병적근본원인.
Objective To study the molecular genetic mechanism of mucopolysaccharidosis type ⅣA(MPS ⅣA), and reveal the relationship between the genotype and phenotype, and provide a basis for prenatal gene diagnosis in the future. Methods A preliminary diagnosis was made by qualitative detection of urinary glycosaminoglycans of the suspected MPS ⅣA proband. Then, mutation detection was performed on the proband and her family members with PCR and direct sequencing of the PCR products. After a novel c.1567T＞G mutation was detected,XspⅠrestriction enzyme digestion and amplification refractory mutation system(ARMS) fast specific identification were established to analyze the sequences of exon 14 in GALNS gene, including 110 randomly selected healthy controls, the proband and other pedigree members. At the same time, bioinformatic approaches for protein secondary, tertiary structure prediction were applied to identify the novel pathologic mutation. Results The proband's urine GAGs test was a weak positive(±),and a c.1567T＞G heterozygous termination codon mutation in exon 14 and a c.374C＞T heterozygous missense mutation in exon 4 were found. The proband was compound heterozygous of the two mutations, so was her younger sister. Her mother was a carrier with only a c.1567T＞G heterozygous mutation in exon 14. Her father had a heterozygous mutation of c.374C>T in exon 4. After XspⅠrestriction enzyme digestion, healthy controls had three bands including 28 bp, 120 bp and 399 bp, while the proband and her mother had four bands consisting of 28 bp, 120 bp,148 bp and 399 bp. For amplification by ARMS specific primers, it was negative for the controls,while it was positive for the proband and the carrier. The results of protein secondary and tertiary structure prediction showed that the c.1567T＞G mutation located in the stop codon, resulted in stop codon (TAG) changing to glutamic acid (GAG), with the peptide chain extending 92 amino acid residues, and secondary and tertiary protein structure change, which were not found in the controls. The result of enzyme assay showed that the activity of GALNS enzyme in the affected child was 8.3 nmol/17h/mg pr, which was obviously lower than the normal value (the normal range is 41.9-92.1 nmol/17h/mg pr). Conclusion These results illustrate that the c.1567 T＞G is a novel pathologic mutation, which is the main cause of the disease in this family.
