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A型血友病乃因第八因子基因異常所引起,利用聚合■鏈反應(polymerase
chain reaction;PCR)及直接核酸序列定位(direct Sequencing)等方
法,可以直接分析病人的第八因子基因. 然而第八因子基因相當巨大,本論
文闡述利用電腦程式設計45對PCR所需的引子(oligonucleotide primers)
來放大第八因子基因的每個特定的exon.接著用單股結構多形性(single
strand conformation polymorphism;SSCP)及雙去氧指印法(dideoxy
fingerprint -ing;ddF)兩種簡單篩檢方法,可偵測出突變的去氧核糖核酸
片段,這兩種方法都是應用單股去氧核糖核酸的特定形狀(distinct
conformation)而偵測出核■酸(nucleotide)的突變. 本研究初步利用
SSCP及ddF鑑定出不正常的 DNA片段,這些DNA片段相對應的exon區域再被
用PCR放大後,定出核酸序列的改變. 在此階段實驗,共分析了87位病人,
其中40位可由SSCP及ddF找出突變型,這些人中5位具輕微至中型症狀,其
餘35位為嚴重型的A型血友病,對於每個病人,本研究均皆找出一個(且僅有
一個)突變型, 而這些突變型中有21個是單一核■酸突變(single
nucleotide mutation),其中8個變成nonsens -e,13個變成missense
codons.另外19位中有16個是減少(delete)或增加了 (insert)1-11個核■
酸,其餘3個卻失掉了一大段基因片段. 利用SSCP,本實驗也檢視8個已被確
定的第八因子多形性(polymorphism),其中3個基因多形性的發生率在中國
人與與西方人不同,這3個分別是codon 1241,codon 1269及 codon
2223,其發生率分別為1.2%,15.7%及97.9%.在本論文進行同時,有實驗報導
指出嚴重型病人中約有50%病人其突變型可能是由於intron22有未明的突
變導致其第八因子mRNA不正常.本研究對於其餘47位病人之中的24位分析
其intron 22. 利用反轉錄-聚合■鏈反應(RT-PCR)的方法,證明17個嚴重
的病人的白血球中具有不正常的第八因子mRNA.綜論,本論文有三個重點:
一.是利用PCR-SSCP,ddF及直接核■酸序列分析找出血友病病人確切的突
變型.二.是證明有38%的嚴重型病人其第八因子基因突變是由於intron
22的未明異常,產生不正常的第八因子mRNA而導致無法製造正常的第八因
子蛋白.三.是利用SSCP分析8種第八因子多形性在中國人的發生率.另外,
本研究至少有二個應用:一.是PCR-SSCP,ddF及直接核■酸序列分析的結果
可應用於病人家屬的帶因者鑑定(carrier detection) 及產前診斷(
prenatal diagnosis).二.是第八因子基因表現(gene expression)及蛋白
結構功能關係(structur -e function relationships)的研究.
The molecular characterization of hemophilia A of Chinese
origin was carried out by the polymerase chain reaction (PCR)
and direct sequencing of patients' factor VIII genes. Forty-
five sets of oligonucleotide primers were designed to allow
specific amplification of individual exons and their flanking
introns of the factor VIII genes.The single strand conformation
polymorphism (SSCP) and dideoxy fingerprinting (ddF) were used
as screening methods to detect mutated DNAs.Both techniques
reveal single base substitutions in a single-stranded DNA as
distinct conformations. The abnormal exon initially identified
by SSCP were subsequently amplified and their nucleotide
sequence determined.A total of 87 patients from different
families were analyzed by PCR-SSCP.Among them,40 cases revealed
mutations in the coding regions of their factor VIII genes.A
single mutational event was found to be associated with each of
the 40 affected individuals.These include 21 cases of single
base mutations,16 cases of deletion or insertion of 1-11
nucleotides,and 3 cases of deletion of large DNA fragments.
Using PCR-SSCP,the frequencies of 8 of the identified factor
VIII polymorphism or silent mutations were also examined among
Chinese.The frequencies for codons 1241,1269 and 2223 were
found to be 1.2%,15.7% and 97.9% respectively, different from
those reported for other populations.As for the 47 cases whose
mutational event was not readily detected by PCR-SSCP ,the
reverse transcriptase combined with PCR (RT-PCR) method was
applied to examine their factor VIII mRNA.In 24 cases
analyzed,17 were found to contain unusual mutations in intron
22 which caused failure in RT-PCR amplification.Our study
presented in this thesis reveals the direct determination of
genetic defects of patients with hemophilia A.This provides a
precise diagnosis for carriers of members in the hemophilic
families.Moreover,the study will be benefit for the study of
factor VIII gene expression and structure-function
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