Candidate Gene Analysis Of Two Familial Hypercholesterolemia Phenotype Families

[Obejective]Familial hypercholesterolemia(FH)is one kind of monogenic autosome dominant inherited disease which always lead to atherosclerosis(AS)and premature coronary heart disease(CAD). Our research choose 2 Chinese FH families.The objective of this study is to find a preliminary screening method that can quickly and effectively analysis the mutation gene of the FH patients. We use the linkage analysis to find the relevance between virulence gene with LDL-R gene and PCSK9 gene, the latter is a new virulence gene with FH.And the linkage analysis we use the new capillary electrophoresis and the new microsatellite . And then we use the DNA nucleotide analysis to locate the gene.[Methods]Our study include three parts: 1.Choosing FH families through the Dutch Lipid Clinic Network in 1999,and we did some coherence check on the proband including ECG, carotid artery ultrasound, ultrasonic cardiogram(UCG), the adenosine burden test and the coronary flow reserve(CFR) .We tested the blood fat of all the families.And then draw the family constellation map for genetics analysis. 2. Using phenol-chloroform to extract DNA and detection of ApoB100 point mutation by PCR and sequencing,and then exclusion the familial ApoB100 defect disease. And next we used linkage analysis to find the relevance between virulence gene with LDL-R gene and PCSK9 gene. According to the LOD ,we can infer the mutation gene initially. 3. 21 segments covered the promotor and the whole 18 LDL-R gene exons were amplified by PCR with the same reaction volume and cycle parameter. The PCR products were sequencinged . And also we amplified PCSK9 gene . The sequence were blasted in the GenBank.[Result]Our results include three parts: 1.We chose 83 family members and there were 11 people in the first family were hyperlipemia . The first proband's brother died of "xanthelasmatosis"in 14 years . We found the first proband has sign of severe atherosclerosis. Electrocardiogram display increased left axis deviation, left ventricle high voltage and V3-V5depressed st segment; Ultrasonic cardiogram display mitral incompetence(midrange), loading test masculine and coronary blood flow store degrade. B ultrasonic display the initial segment endomembrane of carotid artery and vertebral artery thickening;and the initial segment of right subclavian artery display plaque.There were 16 people in the second family were hyperlipemia . The second proband's aunt died of myocardial infarction in 15 years .The electrocardiogram of the second proband display roughly normal. Ultrasonic cardiogram display left ventricular enlargement and mitral reflux. B ultrasonic of the second proband display the initial segment endomembrane of carotid artery and vertebral artery thickening. 2. Linkage analysis found the virulence gene of the first family definitely located in LDL-R gene and maybe located in PCSK9 gene. But linkage analysis in the second family find there is no relation between virulence gene and LDL-R gene or PCSK9 gene . The two proband had no mutation in ApoB100 gene Q3500R,R3531C and R3501W site. 3. We find a novel heterozygosis C→T mutation in the LDL-R extron 2 ,and we call it Q12X. Q12X has been reported in Italian,French and Turks. We certified it is a new morbigenous mutation in china.And we also find the same mutation in the father and the aunts.We found no mutation in LDL-R gene and PCSK9 gene in the second family,so we presume that maybe there is another virulence gene.[Conclusion]1. The probands are dignosised FH and both have severe sign of As.2. The two FH patients have no mutation in ApoB100 gene;3. Linkage analysis find the virulence gene of the first family definitely located in LDL-R gene and maybe located in PCSK9 gene,and a novel heterozygosis T→C mutation in the LDL-R gene was detected in the first proband and his father and his aunts.Q12X is a new morbigenous mutation in China. We have not found PCSK9 gene mutation in the first family. The patient has a high cholesterol level, which probably because the mutation, leading to As progression, it is perhaps reason of CAD occurrence .4. Linkage analysis in the second family find there is no relation between virulence gene and LDL-R gene or PCSK9 gene. There is no discovery in the second family, we presume that maybe there is another virulence gene.We will approach the result in next stage.5. We successfully set up the method to screen the candidate gene through application of a new pattern capillary electrophoresis and microsatellite of new gene.