臺灣博碩士論文加值系統

恰克-馬利-杜斯氏症 (Charcot-Marie-Tooth disease，CMT) 是最常見的遺傳性神經病變，依電生理的特質、遺傳方式、與不同的基因變異可分為許多亞型，其中以CMT1A最常見。CMT1A是自體顯性遺傳的脫髓鞘型神經病變，基因上的變異主要是發生在chromosome 17p11.2-12，內含peripheral myelin protein 22 (PMP22) gene，因為這一段基因發生基因複製 (duplication)，造成患者的DNA之中含有三套的PMP22 gene。同時，若是在同一段基因產生基因缺失 (deletion)，則會造成另一種遺傳性的神經病變，稱之為遺傳性壓力易感性神經病變 (Hereditary neuropathy with liability to pressure palsies，HNPP)。
在診斷方法上，過去主要是依靠臨床的表現，加上家族遺傳史，神經電生理檢查，以及神經切片來做大致上的診斷與分類，然而，上述方法無法提供疾病的基因變異，因此各種分子生物學的方法便孕育而生。
早期所使用的分子生物學的方法各有其優缺點，有的很準確，但是耗時或者比較昂貴，有些方法則敏感性比較不夠。即時定量聚合酶連鎖反應 (real-time quantitative PCR) 具有快速、準確等優點，有相當好的敏感度與特異性，相對節省成本，而且具有很好的再現性，是很好的實驗方法。本實驗即是利用SYBR Green I dye進行即時定量聚合酶連鎖反應的方法來做分子診斷。這個方法的操作簡易、快速，無需設計專用的probes，相對費用較低，很適合拿來作為篩檢之用。利用comparative threshold cycle method來計算PMP22 的基因套數比例 (gene copy number ratio)，我們的方法可以正確的分辨出CMT1A (平均基因套數比例為1.47)、HNPP (平均基因套數比例為0.52)、與control (平均基因套數比例為0.99) 三組檢體，作出正確的分子診斷。
藉由分子診斷，我們可以對患有遺傳性神經病變的患者，或者雖然沒有明確家族史，但是臨床上為脫髓鞘型神經病變的患者作出正確的診斷，讓患者以及家屬可以對疾病有正確的認識以及得到醫療上的協助，對於沒有症狀的家族成員，也可以提供疾病的篩檢與遺傳上的諮詢。

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited peripheral neuropathies. According to the hereditary pattern, clinical manifestations and electrophysiologic findings, CMT could be divided into many subgroups. CMT type 1 is an autosomal dominant demyelinating motor and sensory neuropathy. The most common subtype of CMT type 1 is CMT1A, which is caused by the duplication of a 1.5 Mb region containing the peripheral myelin protein 22 (PMP22) gene on chromosome 17p11.2-12. Hereditary neuropathy with liability to pressure palsies (HNPP), another autosomal dominant peripheral neuropathy, is caused by deletion of the PMP22 gene.
The diagnosis of CMT in the past was mainly dependent on clinical manifestations, family history, electrophysiologic findings and/or sural nerve biopsies. However, these methods could not give information about the underlying genetic defects, so molecular methods were developed for genetic diagnosis. Some molecular diagnostic methods have been used for the diagnosis of CMT. Some methods are accurate, but have the disadvantage of labor-intensive and time-consuming. Some methods are relatively expensive and require specific equipments. We performed real-time quantitative PCR using SYBR Green I dye with our own designed primers for the diagnosis of CMT1A and HNPP. We use comparative threshold cycle method to calculate the estimated PMP22 gene copy number ratio. The estimated PMP22 gene copy number ratio is 1.47 for CMT1A, 0.52 for HNPP and 0.99 for control. Our method could separate the three groups of samples accurately without overlapping.
Real-time quantitative PCR using SYBR Green I dye is a sensitive, specific, accurate and reproducible method for the diagnosis of PMP22 gene duplication or deletion. By this method, we could make diagnosis for patients with CMT1A, HNPP, and other types of hereditary neuropathy and could provide them useful information about medical resources and therapeutic developments of the disease. For asymptomatic and high risk families, this method could also provide genetic screening and counseling.

目錄

中文摘要－－－－－－－－－－－－－－－－－－－－－－5
英文摘要－－－－－－－－－－－－－－－－－－－－－－7
縮寫表－－－－－－－－－－－－－－－－－－－－－－－9
第一章 緒論 －－－－－－－－－－－－－－－－－－－－11
1-1 簡介 －－－－－－－－－－－－－－－－－－－－11
1-2 致病機轉－－－－－－－－－－－－－－－－－－ 15
1-3診斷方法 －－－－－－－－－－－－－－－－－－16
1-3-1傳統診斷法－－－－－－－－－－－－－－－16
1-3-2分子生物學診斷法－－－－－－－－－－－－18
1-4實驗目的 －－－－－－－－－－－－－－－－－－23
第二章 材料與方法－－－－－－－－－－－－－－－－－ 24
2-1檢體之取得與蒐集－－－－－－－－－－－－－－－24
2-2 DNA萃取與測定－－－－－－－－－－－－－－－25
2-3設計PMP22 gene primers －－－－－－－－－－－ 26
2-4測試所設計PMP22 primers之可用性－－－－－－－27
2-5決定即時定量PCR所用之最佳primers濃度 －－－ 27
2-6 Real-time quantitative PCR－－ －－－－－－－－－28
2-7 計算gene copy number－－－－－－－－－－－－ 30
2-8 統計方式－－－－－－－－－－－－－－－－－－31
第三章 結果－－－－－－－－－－－－－－－－－－－－32
第四章 討論－－－－－－－－－－－－－－－－－－－－40
圖－－－－－－－－－－－－－－－－－－－－－－－－ 44
參考文獻 －－－－－－－－－－－－－－－－－－－－－50
附 錄－－－－－－－－－－－－－－－－－－－－－－－59

1.Skre H. Genetic and clinical aspects of Charcot-Marie-Tooth''s disease. Clin Genet 1974;6:98-118.
2.Harding AE, Thomas PK. Genetic aspects of hereditary motor and sensory neuropathy (types I and II). J Med Genet 1980;17:329-36.
3.Berciano J, Combarros O, Calleja J, et al. The application of nerve conduction and clinical studies to genetic counseling in hereditary motor and sensory neuropathy type I. Muscle Nerve 1989;12:302-6.
4.Kaku DA, Parry GJ, Malamut R, et al. Nerve conduction studies in Charcot-Marie-Tooth polyneuropathy associated with a segmental duplication of chromosome 17. Neurology 1993;43:1806-8.
5.Nelis E, Van Broeckhoven C, De Jonghe P, et al. Estimation of the mutation frequencies in Charcot-Marie-Tooth disease type 1 and hereditary neuropathy with liability to pressure palsies: a European collaborative study. Eur J Hum Genet 1996;4:25-33.
6.Choi BO, Kim J, Lee KL, et al. Rapid diagnosis of CMT1A duplications and HNPP deletions by multiplex microsatellite PCR. Mol Cells 2007;23:39-48.
7.Lupski JR, de Oca-Luna RM, Slaugenhaupt S, et al. DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell 1991;66:219-32.
8.Pentao L, Wise CA, Chinault AC, et al. Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit. Nat Genet 1992;2:292-300.
9.Roa BB, Garcia CA, Suter U, et al. Charcot-Marie-Tooth disease type 1A-- Association with a spontaneous point mutation in the PMP22 gene. N Engl J Med 1993;329:96-101.
10.Chance PF, Alderson MK, Leppig KA, et al. DNA deletion associated with hereditary neuropathy with liability to pressure palsies. Cell 1993;72:143-51.
11.Andersson PB, Yuen E, Parko K, et al. Electrodiagnostic features of hereditary neuropathy with liability to pressure palsies. Neurology 2000;54:40-4.
12.Infante J, Garcia A, Combarros O, et al. Diagnostic strategy for familial and sporadic cases of neuropathy associated with 17p11.2 deletion. Muscle Nerve 2001;24:1149-55.
13.Pareyson D, Scaioli V, Taroni F, et al. Phenotypic heterogeneity in hereditary neuropathy with liability to pressure palsies associated with chromosome 17p11.2-12 deletion. Neurology 1996;46:1133-7.
14.Nicholson GA, Valentijn LJ, Cherryson AK, et al. A frame shift mutation in the PMP22 gene in hereditary neuropathy with liability to pressure palsies. Nat Genet 1994;6:263-6.
15.Hoogendijk JE, Hensels GW, Zorn I, et al. The duplication in Charcot-Marie-Tooth disease type 1A spans at least 1100 kb on chromosome 17p11.2. Hum Genet 1991;88:215-8.
16.Raeymaekers P, Timmerman V, Nelis E, et al. Duplication in chromosome 17p11.2 in Charcot-Marie-Tooth neuropathy type 1a (CMT 1a). The HMSN Collaborative Research Group. Neuromuscul Disord 1991;1:93-7.
17.Hayasaka K, Himoro M, Sato W, et al. Charcot-Marie-Tooth neuropathy type 1B is associated with mutations of the myelin P0 gene. Nat Genet 1993;5:31-4.
18.Warner LE, Mancias P, Butler IJ, et al. Mutations in the early growth response 2 (EGR2) gene are associated with hereditary myelinopathies. Nat Genet 1998;18:382-4.
19.Kamholz J, Menichella D, Jani A, et al. Charcot-Marie-Tooth disease type 1: molecular pathogenesis to gene therapy. Brain 2000;123:222-33.
20.Gow A, Friedrich VL, Jr., Lazzarini RA. Intracellular transport and sorting of the oligodendrocyte transmembrane proteolipid protein. J Neurosci Res 1994;37:563-73.
21.Gow A, Lazzarini RA. A cellular mechanism governing the severity of Pelizaeus-Merzbacher disease. Nat Genet 1996;13:422-8.
22.Naef R, Adlkofer K, Lescher B, et al. Aberrant protein trafficking in Trembler suggests a disease mechanism for hereditary human peripheral neuropathies. Mol Cell Neurosci 1997;9:13-25.
23.Tobler AR, Notterpek L, Naef R, et al. Transport of Trembler-J mutant peripheral myelin protein 22 is blocked in the intermediate compartment and affects the transport of the wild-type protein by direct interaction. J Neurosci 1999;19:2027-36.
24.Lawson VH, Gordon Smith A, Bromberg MB. Assessment of axonal loss in Charcot-Marie-Tooth neuropathies. Exp Neurol 2003;184:753-7.
25.Meyer Zu Horste G, Nave KA. Animal models of inherited neuropathies. Curr Opin Neurol 2006;19:464-73.
26.Shy ME, Blake J, Krajewski K, et al. Reliability and validity of the CMT neuropathy score as a measure of disability. Neurology 2005;64:1209-14.
27.Cornblath DR, Chaudhry V, Carter K, et al. Total neuropathy score: validation and reliability study. Neurology 1999;53:1660-4.
28.Holland NR, Stocks A, Hauer P, et al. Intraepidermal nerve fiber density in patients with painful sensory neuropathy. Neurology 1997;48:708-11.
29.Kennedy WR, Wendelschafer-Crabb G. The innervation of human epidermis. J Neurol Sci 1993;115:184-90.
30.Kennedy WR, Wendelschafer-Crabb G, Johnson T. Quantitation of epidermal nerves in diabetic neuropathy. Neurology 1996;47:1042-8.
31.Polydefkis M, Hauer P, Griffin JW, et al. Skin biopsy as a tool to assess distal small fiber innervation in diabetic neuropathy. Diabetes Technol Ther 2001;3:23-8.
32.Polydefkis M, Yiannoutsos CT, Cohen BA, et al. Reduced intraepidermal nerve fiber density in HIV-associated sensory neuropathy. Neurology 2002;58:115-9.
33.Holland NR, Crawford TO, Hauer P, et al. Small-fiber sensory neuropathies: clinical course and neuropathology of idiopathic cases. Ann Neurol 1998;44:47-59.
34.Nolano M, Provitera V, Crisci C, et al. Quantification of myelinated endings and mechanoreceptors in human digital skin. Ann Neurol 2003;54:197-205.
35.Ceuterick-de Groote C, De Jonghe P, Timmerman V, et al. Infantile demyelinating neuropathy associated with a de novo point mutation on Ser72 in PMP22 and basal lamina onion bulbs in skin biopsy. Pathol Res Pract 2001;197:193-8.
36.Li J, Bai Y, Ghandour K, et al. Skin biopsies in myelin-related neuropathies: bringing molecular pathology to the bedside. Brain 2005;128:1168-77.
37.MacMillan JC, Upadhyaya M, Harper PS. Charcot-Marie-Tooth disease type 1a (CMT1a): evidence for trisomy of the region p11.2 of chromosome 17 in south Wales families. J Med Genet 1992;29:12-3.
38.Raeymaekers P, Timmerman V, Nelis E, et al. Estimation of the size of the chromosome 17p11.2 duplication in Charcot-Marie-Tooth neuropathy type 1a (CMT1a). HMSN Collaborative Research Group. J Med Genet 1992;29:5-11.
39.Chance PF, Abbas N, Lensch MW, et al. Two autosomal dominant neuropathies result from reciprocal DNA duplication/deletion of a region on chromosome 17. Hum Mol Genet 1994;3:223-8.
40.Timmerman V, Lofgren A, Le Guern E, et al. Molecular genetic analysis of the 17p11.2 region in patients with hereditary neuropathy with liability to pressure palsies (HNPP). Hum Genet 1996;97:26-34.
41.Chen KL, Wang YL, Dodson LA, et al. Normalized Southern Hybridization to Enhance Testing for Charcot-Marie-Tooth Disease, Type 1A. Mol Diagn 1996;1:65-71.
42.Hoogendijk JE, Hensels GW, Gabreels-Festen AA, et al. De-novo mutation in hereditary motor and sensory neuropathy type I. Lancet 1992;339:1081-2.
43.Hoogendijk JE, Janssen EA, Gabreels-Festen AA, et al. Allelic heterogeneity in hereditary motor and sensory neuropathy type Ia (Charcot-Marie-Tooth disease type 1a). Neurology 1993;43:1010-5.
44.Ikegami T, Ikeda H, Chance PF, et al. Facilitated diagnosis of CMT1A duplication in chromosome 17p11.2-12: analysis with a CMT1A-REP repeat probe and photostimulated luminescence imaging. Hum Mutat 1997;9:563-6.
45.Aarskog NK, Vedeler CA. Real-time quantitative polymerase chain reaction. A new method that detects both the peripheral myelin protein 22 duplication in Charcot-Marie-Tooth type 1A disease and the peripheral myelin protein 22 deletion in hereditary neuropathy with liability to pressure palsies. Hum Genet 2000;107:494-8.
46.Roa BB, Greenberg F, Gunaratne P, et al. Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1 neuropathy. Hum Genet 1996;97:642-9.
47.Shaffer LG, Kennedy GM, Spikes AS, et al. Diagnosis of CMT1A duplications and HNPP deletions by interphase FISH: implications for testing in the cytogenetics laboratory. Am J Med Genet 1997;69:325-31.
48.Lopes J, LeGuern E, Gouider R, et al. Recombination hot spot in a 3.2-kb region of the Charcot-Marie-Tooth type 1A repeat sequences: new tools for molecular diagnosis of hereditary neuropathy with liability to pressure palsies and of Charcot-Marie-Tooth type 1A. French CMT Collaborative Research Group. Am J Hum Genet 1996;58:1223-30.
49.Reiter LT, Murakami T, Koeuth T, et al. A recombination hotspot responsible for two inherited peripheral neuropathies is located near a mariner transposon-like element. Nat Genet 1996;12:288-97.
50.Timmerman V, Rautenstrauss B, Reiter LT, et al. Detection of the CMT1A/HNPP recombination hotspot in unrelated patients of European descent. J Med Genet 1997;34:43-9.
51.Yamamoto M, Yasuda T, Hayasaka K, et al. Locations of crossover breakpoints within the CMT1A-REP repeat in Japanese patients with CMT1A and HNPP. Hum Genet 1997;99:151-4.
52.Chang JG, Jong YJ, Wang WP, et al. Rapid detection of a recombinant hotspot associated with Charcot-Marie-Tooth disease type IA duplication by a PCR-based DNA test. Clin Chem 1998;44:270-4.
53.Blair IP, Kennerson ML, Nicholson GA. Detection of Charcot-Marie-Tooth type 1A duplication by the polymerase chain reaction. Clin Chem 1995;41:1105-8.
54.Wise CA, Garcia CA, Davis SN, et al. Molecular analyses of unrelated Charcot-Marie-Tooth (CMT) disease patients suggest a high frequency of the CMTIA duplication. Am J Hum Genet 1993;53:853-63.
55.Latour P, Boutrand L, Levy N, et al. Polymorphic short tandem repeats for diagnosis of the Charcot-Marie-Tooth 1A duplication. Clin Chem 2001;47:829-37.
56.Chen SR, Lin KP, Kuo HC, et al. Comparison of two PCR-based molecular methods in the diagnosis of CMT 1A and HNPP diseases in Chinese. Clin Neurol Neurosurg 2008;110:466-71.
57.Su YN, Hung CC, Li H, et al. Quantitative analysis of SMN1 and SMN2 genes based on DHPLC: a highly efficient and reliable carrier-screening test. Hum Mutat 2005;25:460-7.
58.Kumar J, Kumar A, Das SK, et al. Detection of differential gene copy number using denaturing high performance liquid chromatography. J Biochem Biophys Methods 2005;64:226-34.
59.Lin CY, Su YN, Lee CN, et al. A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay. J Hum Genet 2006;51:227-35.
60.Naimi M, Tardieu S, Depienne C, et al. Detection of genomic rearrangements by DHPLC: a prospective study of 90 patients with inherited peripheral neuropathies associated with 17p11.2 rearrangements. Am J Med Genet A 2005;136:136-9.
61.Hung CC, Lee CN, Lin CY, et al. Identification of deletion and duplication genotypes of the PMP22 gene using PCR-RFLP, competitive multiplex PCR, and multiplex ligation-dependent probe amplification: a comparison. Electrophoresis 2008;29:618-25.
62.Schouten JP, McElgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57.
63.Heid CA, Stevens J, Livak KJ, et al. Real time quantitative PCR. Genome Res 1996;6:986-94.
64.Lie YS, Petropoulos CJ. Advances in quantitative PCR technology: 5'' nuclease assays. Curr Opin Biotechnol 1998;9:43-8.
65.Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med 1998;36:255-69.
66.Kim SW, Lee KS, Jin HS, et al. Rapid detection of duplication/deletion of the PMP22 gene in patients with Charcot-Marie-Tooth disease Type 1A and hereditary neuropathy with liability to pressure palsy by real-time quantitative PCR using SYBR Green I dye. J Korean Med Sci 2003;18:727-32.
67.Choi JR, Lee WH, Sunwoo IN, et al. Effectiveness of real-time quantitative PCR compare to repeat PCR for the diagnosis of Charcot-Marie-Tooth type 1A and hereditary neuropathy with liability to pressure palsies. Yonsei Med J 2005;46:347-52.
68.Livak KJ. Comparative Ct method. ABI Prism 7700 Sequence Detection System. In: User Bulletin No 2, PE Applied Bio-systems; 1997.