摘要
家族性高膽固醇血症(FH)為一體染色體顯性遺傳疾病，患者因LDLR基因突變而導致血漿中LDL膽固醇值過高，易發展出早發性的冠狀心臟病。本論文利用multiplex PCR、SSCP分析及DNA定序等技術，檢示臺灣36位高血脂患者及2位FH患者的LDLR基因變異，結果發現C308Y突變(即表現子7上第308個胺基酸密碼發生TGCTAC的改變)及Del exon 6-8突變(即缺失表現子6~8的123個胺基酸)。本研究對此2種變異及先前本研究室所發現的W-18X、D69N、I402V、A410T、I602V、A696G等變異作進一步探討。D102家族的DdeI檢測試驗顯示，患者並未將W-18X遺傳給子代。A133家族MfeI檢測試驗顯示，患者的第三個兒子及一位孫子均為D69N的異型合子。進一步的多型性單套型分析顯示，患者L41與A133的D69N可能具相同的起源。D63家族的AccI檢測試驗顯示，患者D63為C308Y的同型合子，其父母親、二哥及兩位姐姐則皆為C308Y的異型合子。此家族中C308Y與高血脂質有明顯的共分離。單套型分析顯示D63的父母為近親婚配，且C308Y單套型與香港中國人的C308Y單套型相同，推測可能是中國人常見的突變。I602V於三位高血脂患者與2位正常人中亦被發現，故I602V應為一未報導過的多型性。D162家族的PstI檢測試驗顯示，患者的A696G並未遺傳給子代。由於A696G在正常人中並未被發現，故可能為未報導過的突變或罕見的變異。患者D254的Del exon 6-8係因介入子5與8的Alu 序列發生同源性重組所致。D254的父母亦為近親婚配，所蒐集的家族成員中有15名帶有此缺失突變。在此家族中，此突變與高血脂質明顯的共分離。在轉移的COS-7細胞中，野生型的LDLR cDNA重組質體表現160 kDa成熟蛋白及少量的120 kDa前驅蛋白。含D69N突變的重組質體表現的LDLR mRNA量明顯的較低(野生型的40%)，且出現中間型的前驅蛋白。含C308Y、I402T及A410T突變的重組質體所表現的mRNA量與野生型者相近，但所表現的成熟蛋白均較野生型者少。含A696G的mRNA及成熟蛋白含量則均與野生型者相近。

ABSTRACT
FH is an autosomal dominant disorder of lipoprotein metabolism, primarily caused by mutation in the LDLR gene. FH patients have elevated plasma LDL cholesterol and generally developed coronary heart disease. The study used the multiplex PCR, SSCP and DNA sequence analyses to examine the LDLR gene of 36 hyperlipidemic patients and 2 FH patients in Taiwan. A point mutation (C-to-A transversion in codon 308) and a deletion mutation Del exons 6-8 (deletion of 123 amino acids on exons 6-8) were found. The two mutations and six previously identified mutations (W-18X, D69N, I402T, A410T, I602V, A696G) were further characterized. By PCR and DdeI restriction analysis, none of the daughters inherited the W-18X mutation from D102. By MfeI restriction analysis, the D69N mutation was found in A133's son and grandson. The results of haplotype analysis suggest that the D69N alleles of A133 and L41 are likely identical by descent. Patient D63 was homozygous for the C308Y mutation. By AccI restriction analysis, both her parents and three of her siblings also carry the mutation. The mutation cosegregated with hypercholesterolemia in this family. The same haplotype of two C308Y alleles in D63 was resulted from consanguineous marriage. Comparison of this haplotype with that reported in two Chinese patients in Hong Kong suggests that the C308Y mutation arose in a common ancestor of Chinese origin. The novel I602V polymorphism was found in 3 hyperlipidemic patients and 2 normolipidemic controls. By PstI restriction analysis, the D162's son did not inherit the mutation A696G. Due to not seen in normal controls, A696G may be a novel mutation or a rare variant. The deletion Del exons 6-8 was caused by intrastrand recombination event between Alu sequences in introns 5 and 8. The same haplotype of two Del exons 6-8 alleles in D254 was resulted from consanguineous marriage. Fifteen of the recruited family members also carry the deletion. The deletion co-segregated with hypercholesterolemia in this family. In transfected COS-7 cells, wild type LDLR cDNA expressed 160 kDa mature and 120 kDa precursor proteins. An apparent reduction in LDLR mRNA level (40% of wild type) and a novel precursor protein were seen in D69N transfected cells. Although normal amount of LDLR mRNA was seen with the C308Y, I402T and A410T mutations, the amount of mature protein was less than that of wild type. The LDLR mRNA and protein levels were close to those of wild type in A696G transfected cells.

伍、參考文獻
Berge KE, Tian H, Graf GA, Yu L, Grishin NV, Schultz J, Kwiterovich P, Shan B, Barnes R, Hobbs HH. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science 2000;290:1771-75.
Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986;232:34-47.
Cai HJ, Fan LM, Huang MG, Chen XY, Liu GQ, Chen P. Homozygous familial hypercholesterolemia patients in China. Atherosclerosis 1985;57:303-12.
Cotran RS, Kumar V, Robbins SL. Robbins pathologic basis of disease, 4th ed. W.B. Saunders Company. 1989:137-143.
Day INM, Haddad L, O’Dell SD, Day LB, Whittall RA, Humphries SE. Identification of a common low density lipoprotein receptor mutation (R329X) in the south of England: complete linkage disequilibrium with an allele of microsatellite D19S394. J Med Genet 1997a;34:111-6.
Day INM, Whittall RA, O’Dell SD, Haddad L, Bolla MK, Gudnason V, Humphries SE. Spectrum of LDL recepter gene mutations in heterozygous familial hypercholesterolemia. Hum Mutat 1997b;10:116-27.
Ekstrom U, Abrahamson M, Sveger T, Lombardi P, Nilsson-Ehle P. An efficient screening procedure detecting six novel mutations in the LDL receptor gene in Swedish children with hypercholesterolemia. Hum Genet 1995;96:147-50.
Ekstrom U, Abrahamson M, Sveger T, Sun XM, Soutar AK, Nilsson-Ehle P. Expression of an LDL recepter allele with two different mutations (E256K and I402T). Mol Pathol 2000 Feb;53(1):31-6.
Garcia CK, Wilund K, Arca M, Zuliani G, Fellin R, Maioli M, Calandra S, Bertolin S, Cohen JC, Hobb HH. Autosomal recessive hypercholesterol-emia caused by mutations in putative LDL recepter adaptor protein. Scince 2001;292:1394-8.
Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolemia. In: Scriver ER, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited diseases, 6th ed. New York: McGraw-Hill Book Co., 1995:1981-2030.
Hattori H, Nagano M, Iwasaki T, Egashira T, Homma Y. New missense and frameshit mutations in the LDL recepter gene causing Janpanese familial hypercholesterolemia. Atherosclersis 1997;134:68.
Heath KE, Gahan M, Whittall RA, Humphries SE. Low-density lipoprotein receptor gene (LDLR) world-wide website in familial hypercholesterol-emia: update, new features and mutation analysis. Atherosclerosis 2001;154:243-6.
Hobbs HH, Russel DW, Brown MS, Goldstein JL. The LDL receptor locus in familial hypercholesterolemia: mutation analysis of a membrane protein. Annu Rev Genet 1990;24:133-70.
Hobbs HH, Brown MS, Goldstein JL. Molecular genetics of the LDL receptor gene in familial hypercholesterolemia. Hum Mutat 1992;1:445-66.
Innerarity TL, Mahley RW, Weisgraber KH, Bersot TP, Krauss RM, Vega GL, Grundy SM, Friedl W, Davignon J, McCarthy BJ. Familial defective apolipoprotein B-100: a mutation of apolipoprotein B that causes hypercholesterolemia. J Lipid Res 1990;31:1337-49.
Khoo KL, van Acker P, Defesche JC, Tan H, van de Kerkhof L, Heijnen-van Eijk SJ, Kastelein JJP, Deslypere JP. Low-density lipoprotein recepter gene mutations in Southeast Asian population with familial hypercholesterolemia. Clin Genet 2000;58:98-105.
Kotze MJ, Langenhoven E, Warnich L, du Plessis L, Retief AE. The molecular basis and diagnosis of familial hypercholesterolemia in South African Afrikaners. Ann Hum Genet 1991;55:115-21.
Lehninger, AL, Nelson DL, Cox MM. Principles of Biochemistry. 2th ed. New York: Worth Publisher. 1993:479-505.
Lehrman MA, Schneider WJ, Sudhof TC, Brown MS, Goldstein JL, Russell DW. Mutation in LDL receptor: Alu-Alu recombination deletes exons encoding transmembrane and cytoplasmic domain. Science 1985;227:140-6.
Lehrman MA, Russell DW, Goldstein JL, Brown MS. Exon-Alu recombination deletes 5 kilobases from low density lipoprotein receptor gene, producing a null phenotype in familial hypercholesterolemia. Proc Natl Acad Sci USA 1986;832:3679-83.
Lehrman MA, Goldstein JL, Russell DW, Brown MS. Duplication of seven exons in LDL receptor gene caused by Alu-Alu recombination in a subject with familial hypercholesterolemia. Cell 1987a;48:827-35.
Lehrman MA, Schneider WJ, Brown MS, Davis CG, Elhammer A, Russell DW, Goldstein JL. The Lebanese allele at the low density lipoprotein recepter locous. J Biol Chem 1987b;262:401-10.
Lehrman MA, Russell DW, Goldstein JL, Brown MS. Alu-Alu recombination deletes splice acceptor sites and produces secreted LDL receptor in a subject with familial hypercholesterolemia. J Biol Chem 1987c;262:3354-61.
Leitersdorf E, Tobin EJ, Davignon J, Hobbs HH. Common low-density lipoprotein receptor mutations in the French Canadian population. J Clin Invest 1990;85:1014-23.
Lindgren, V., Lusky, K. L., Russell, D.W., Francke, U. Human genes involved in cholesterol metabolism: chromosomal mapping of the loci for the low density lipoprotein recepter and 3-hydroxy-3-methylglutaryl-coenzyme A reductase with cDNA probes. Proc Natl Acad Sci USA 1985;82:8567-71.
Maruyama T, Miyake Y, Tajima S, Harada-Shiba M, Yamamura T, Tsushima M, Kishino B, Horiguchi Y, Funahashi T, Matsuzawa Y, Yamamoto A. Common mutations in the low-density-lipoprotein-receptor gene causing familial hypercholesterolemia in the Japanese population. Arterioscler Thromb 1995;15:1713-8.
Mak YT, Pang CP, Tomlinson B, Zhang J, Chan YS, Mak TWL, Masarei JRL. Mutations in the low-density lipoprotein receptor gene in Chinese familial hypercholesterolemia patients. Arterioscler Thromb Vasc Biol 1998a;18:1600-5.
Mak YT, Zhang J, Chan YS, Mak TWL, Tomlinson B, Masarei JRL, Pang CP. Possible common mutations in the Low density lipoprotein receptor gene in Chinese. Hum Mutat supplement 1998b;1:S310-3.
Myant, N.B. Familial defective apolipoprotein B-100: a review, including some comparisons with familial hypercholesterolaemia. Atherosclerosis 1993;104:1-18.
Pimstone SN, Sun XM, du Souich C, Frohlich JJ, Hayden MR, Soutar AK. Phenotypic variation in heterozygous familial hypercholesterolemia: a comparison of Chinese patients with the same or similar mutations in the LDL receptor gene in China or Canada. Arterioscler Thromb Vasc Biol 1998;18:309-315.
Report of the National Cholesterol Education Program Expert Panel on Detection, Evalution, and Treatment of High Blood Cholesterol in Adults. The Expert Panel, Arch Intern Med 1988;148:36-69.
Sambrook EH, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press. 1989:E5, 1.21-1.25, 7.37-7.52.
Schmid CW, Jelinek WR. The Alu family of dispersed repetitive sequences. Science 1982;216:1065-70.
Sudhof TC, Goldstein JL, Brown MS, Russell DW. The LDL receptor gene: a mosaic of exons shared with different proteins. Science 1985;228:815-22.
Sun XM, Patel DD, Webb JC, Knight BL, Fan LM, Cai HJ, Soutar AK. Familial hypercholesterolemia in China: identification of mutations in the LDL-receptor gene that result in a receptor-negative phenotype. Arterioscler Thromb Vasc Biol 1994;14:85-94.
Tai DY, Pan JP, Lee-Chen GJ. Identification and haplotype analysis of apolipoprotein B-100 Arg3500Trp mutation in hyperlipidemic Chinese. Clin Chem 1998;44:1659-65.
Teng YN, Pan JP, Chou SC, Tai DY, Lee-Chen GJ. Familial defective apolipoprotein B-100: detection and haplotype analysis of the Arg3500Gln mutation in hyperlipidemic Chinese. Atherosclerosis 2000;152:385-90.
Thompson, G.R. A Handbook of Hyperlipidemia. London: Current Science Ltd. 1989.
Vega GL, Grundy SM. In vivo evidence for reduced binding of low density lipoproteins to recepters as a cause of primary moderate hypercholester-olemia. J Clin Invest 1986;78:1410-14.
Yamamoto T, Davis CG, Brown MS, Schneider WJ, Casey ML, Goldstein JL, Russell DW. 1984. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell 1984;39:27-38.
Zuliani G, Hobbs HH. Dinucleotide repeat polymorphism at the 3' end of the LDL recepter gene. Nucleic Acid Res 1990;18:4300.