应用改良连锁分析初筛家族性高胆固醇血症的候选基因

Using Improved Linkage Analysis to Screen Candidate Gene of Familial Hypercholesterolemia

目的应用改良连锁分析方法对2个纯合子家族性高胆固醇血症(FH)家系进行候选基因初筛,为后期测序明确方向,早期发现致病基因。方法 2010年6月和2010年7月首都医科大学附属北京安贞医院收治2例FH先证者,所有家系成员进行血脂测定、心电图、超声心动图及颈动脉超声检查,记录家系成员的其他临床资料(年龄、性别、发病年龄、病程、家族史及治疗情况)。选择新的微卫星位点和毛细管电泳仪进行连锁分析初筛,高度连锁者进一步测序明确致病基因。结果先症者1心电图示左心室肥厚;先症者2心电图大致正常。先证者1超声心动图示主动脉瓣反流;先证者2超声心动图示左房室瓣反流。先证者1颈动脉超声示颈动脉内中膜增厚,颈动脉内斑块形成;先证者2颈动脉超声示颈动脉内中膜增厚。家系1中胆固醇增高者总胆固醇(TC)水平〔(6.98±1.99)mmol/L〕高于血脂正常者〔(3.20±1.02)mmol/L〕(t=7.023,P<0.001);胆固醇增高者低密度脂蛋白胆固醇(LDL-C)水平〔(3.02±2.26)mmol/L〕高于血脂正常者〔(1.98±0.93)mmol/L〕(t=3.497,P=0.004)。家系2中胆固醇增高者TC水平〔(8.12±3.65)mmol/L〕高于血脂正常者〔(4.37±1.01)mmol/L〕(t=4.355,P=0.001);胆固醇增高者LDL-C水平〔(5.72±3.92)mmol/L〕高于血脂正常者〔(2.72±0.62)mmol/L〕(t=3.293,P=0.005)。改良连锁分析发现先症者1的致病基因与低密度脂蛋白受体(LDL-R)基因高度连锁,进一步核苷酸序列分析发现该患者LDL-R突变是中国罕见致病突变。先证者2的致病基因与已知致病基因均不连锁,推测可能存在第4种新的未知致病基因。结论 FH应用改良连锁分析方法成功发现1例中国罕见突变,推测可能还有新的未知致病基因存在。

Objective Improved linkage analysis was applied to screen candidate gene of two homozygous familial hypercholesterolemia families,providing a clear subsequent sequencing direction for early detection of pathogenic gene. Methods 2 probands of FH were enrolled in Beijing Anzhen Hospital,Capital Medical University in June 2010 and July 2010. All the family members underwent serum lipid determination,electrocardiogram,echocardiogram and carotid artery ultrasound,and other clinical informations（age,gender,age of onset,course of disease,family history and treatment）of family memberswere recorded. We selected the new microsatellite markers and capillary electrophoresis for linkage analysis of screening,and then sequenced the high linkaged one to confirm the pathogenic gene. Results The ECG of proband 1 showed left ventricular hypertrophy. The ECG of proband 2 was generally normal. The echocardiography of proband 1 showed aortic regurgitationwhile that of proband 2 showed left atrioventricular valve regurgitation. The ultrasonography of carotid artery in proband 1 found intima -media thickening and plaque formation,while that in proband 2 found carotid intima - media thickening. In family 1 the level of TC〔（6. 98 ± 1. 99） mmol/ L〕in subjects with high cholesterol level was significantly higher than that〔（3. 20 ± 1. 02） mmol/ L〕in subjects with normal blood lipid level（ t = 7. 023,P 〈 0. 001）,and the level of LDL - C〔（3. 02 ± 2. 26） mmol/ L〕in subjects with high cholesterol level was significantly higher than that〔（1. 98 ± 0. 93）mmol/ L〕in subjects with normal blood lipid level（t = 3. 497,P = 0. 004）. In family 2 the level of TC〔（8. 12 ± 3. 65）mmol/ L〕in subjects with high＆nbsp;cholesterol level was significantly higher than that〔（4. 37 ± 1. 01） mmol/ L〕in subjects with normal blood lipid level（ t= 4. 355,P = 0. 001）,and the level of LDL - C〔（5. 72 ± 3. 92）mmol/ L〕in subjects with high cholesterol level was higher than that〔（2. 72 ± 0. 62） mmol/ L〕in subjects with normal blood lipid level（ t = 3. 293,P = 0. 005）. Improved linkage analysis found that pathogenic gene in proband 1 was linked to LDL - R gene,further nucleotide sequence analysis showed that the LDL - R mutation of the patient was a rare pathogenic mutation in China. Pathogenic genes in proband 2 were not linked to any known genes,which suggested that the fourth new unknown pathogenic gene might exist. Conclusion The improved linkage analysis for early screening of FH pathogenic gene can save manpower,financial and material resources. The newfound rare mutation in China suggests that a new unknown gene may exist.