CAJ | 학술논문

为了研究粘多糖贮积症Ⅱ型(MPSⅡ)患者发病的分子遗传学机制,以便为今后的产前基因诊断等创造必要的前提条件,文章先采用尿糖胺聚糖(GAGs)定性检测法对疑似MPSⅡ的先证者进行初诊,然后采用PCR、PCR产物直接测序法对先证者及其家系成员进行突变检测.在检出IDS基因c.876del2新突变后,对随机采集的120例正常对照和其他非Ⅱ型MPS患者包括MPS Ⅰ,Ⅳ,Ⅵ三型的病人共15例的IDS基因exon 6进行序列分析,同时采用不同物种突变点序列的保守性分析法,以及直接测定患儿及其家庭相关成员IDS酶活性的方法对该新突变进行致病性分析.结果显示:先证者尿检呈强阳性(GAGs+++);其IDS基因exon 6编码区内存在c.876-877 del TC新缺失突变,为半合子突变,而其母、其姐为杂合突变;正常对照和其他非Ⅱ型MPS患者的IDS基因exon 6的检测结果均未发现该突变;不同物种氨基酸序列的同源性比对显示:c.876-877 del TC突变所在的位置即P.292-293的苯丙氨酸(F)谷氨酰胺(Q)高度保守;酶活性测定的结果显示:先证者的IDS酶活性仅为2.3 nmoF4 h/mL,大大低于正常值,而其父的为641.9 nmol/4 h/mL,其母的血浆酶活性为95.8 nmol/4h/mL,其姐的为103.2 nmol/4 h/mL.说明所发现的c.876-877 del TC缺失移码突变是一种新的病理性突变,是该MPSⅡ患儿发病的根本内因.
위료연구점다당저적증Ⅱ형(MPSⅡ)환자발병적분자유전학궤제,이편위금후적산전기인진단등창조필요적전제조건,문장선채용뇨당알취당(GAGs)정성검측법대의사MPSⅡ적선증자진행초진,연후채용PCR、PCR산물직접측서법대선증자급기가계성원진행돌변검측.재검출IDS기인c.876del2신돌변후,대수궤채집적120례정상대조화기타비Ⅱ형MPS환자포괄MPS Ⅰ,Ⅳ,Ⅵ삼형적병인공15례적IDS기인exon 6진행서렬분석,동시채용불동물충돌변점서렬적보수성분석법,이급직접측정환인급기가정상관성원IDS매활성적방법대해신돌변진행치병성분석.결과현시:선증자뇨검정강양성(GAGs+++);기IDS기인exon 6편마구내존재c.876-877 del TC신결실돌변,위반합자돌변,이기모、기저위잡합돌변;정상대조화기타비Ⅱ형MPS환자적IDS기인exon 6적검측결과균미발현해돌변;불동물충안기산서렬적동원성비대현시:c.876-877 del TC돌변소재적위치즉P.292-293적분병안산(F)곡안선알(Q)고도보수;매활성측정적결과현시:선증자적IDS매활성부위2.3 nmoF4 h/mL,대대저우정상치,이기부적위641.9 nmol/4 h/mL,기모적혈장매활성위95.8 nmol/4h/mL,기저적위103.2 nmol/4 h/mL.설명소발현적c.876-877 del TC결실이마돌변시일충신적병이성돌변,시해MPSⅡ환인발병적근본내인.
The purpose of this study was to understand the molecular genetic mechanism of mucopolysaccharidosis type II (MPS II) and to provide a prerequisite for future prenatal gene diagnosis. A preliminary diagnosis was made by qualitative detection of Urinary Glycosaminoglycans of the suspected MPS II proband. Then, mutation detection was performed on the proband and his family members with PCR and direct sequencing of PCR products. After the novel mutation of c.876 del 2 in IDS gene was detected, sequence analysis was performed on exon 6 of IDS gene of the 135 cases, which consisted of 120 randomly selected normal controls, and other 15 patients with MPS I, IV, and VI other than MPS II. Besides, the patho-genicity of the novel mutation was analyzed with the following 2 methods: conservative analysis of the sequence of mutation spots of different species and the direct test of the IDS enzyme activity of the patient and his relative family members. The result of uroscopy of the proband was Strong positive (GAGs +++). There was a novel deletion mutation of c.876-877 del TC in the coding region of exon 6 of IDS gene, which was a hemizygous mutation. However, the mutation of his mother and sister was a heterozygous mutation. Detection of the exon 6 of IDS gene showed that the mutation was not found among normal controls and other patients with MPS I, IV, and VI other than MPS II. Homology comparison of amino acid sequences from different species showed that the phenylalanine (F) glutamine (Q) of the mutation site of c. 876-877 del TC located in p.292-293 was highly conserved. The activity of IDS enzyme of the proband was only 2.3 nmol/4 h/mL, which was much lower than normal; but the activity of IDS enzyme of his father, mother and sister was 641.9 nmol/4 h/mL, 95.8 nmol/4h/mL and 103.2 nmol/4h/mL, respectively. These results illustrated that the deletion and frame-shift mutation of c.876-877 del TC detected was a novel pathologic mutation, which was the underlying cause of MPS II of this patient.