Establishment of Genetic Diagnostic Techniques and Gene Dosage Analytical Systems in Genetic Disorders

• Main Authors: Chia-Cheng Hung, 洪加政
• Other Authors: Win-Li Lin
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/67978278310138492058

博士 === 國立臺灣大學 === 醫學工程學研究所 === 95 === As we are now entering the post-genomic era, there are a number of new research trends. One trend will interrogate the influence of common genetic variants within the human genome. These common variants, which are often referred as polymorphisms, have already been proven to play a role in genomic disease, drug toxicity, and general pharmaceutical efficacy. Genetic polymorphisms (particularly single-nucleotide polymorphisms, SNPs, which account for ~90% of common genetic variants) are currently used within various association studies for disease research. Another trend will be an increase in the number of disease is that the various genetic mutations within a functional pathway and reveal their linkages to the diseases. This genetic various on the functional regions within the genome, including trinucleotide repeat expansions, associated with genetic disorders caused by misfolded protein. The third trend is to provide clinical genetic diagnosis by utilizing the available SNP and mutation analytical tool for earlier disease prevention, prenatal diagnosis and health care. In this application, accuracy, speed, automation, reliability, affordability, and flexibility, as well as the ability to detect both known and unknown mutations will be of great importance. These three trends alone demonstrate the post-genomic era’s requirements for genetic analysis technologies that can provide high degrees of sample throughput without sacrificing sensitivity, as well as high levels of automation without sacrificing flexibility. To address these needs, we present a new nucleic acid analysis technology, including denaturing high-performance liquid chromatography (DHPLC), capillary electrophoresis (CE), melting curve analysis and MLPA. The DHPLC have proven to be a promising tool for nucleic acids separation. The success of the DHPLC approach to genetic analyses is demonstrated in its applications by genetic mutation and polymorphism discovery and screening in many commercial clinical diseases diagnosis. We develop two DHPLC methods including heteroduplex analysis and single base primer extension, to optimize the efficiency in genetic diagnosis for analysis of genes with unknown mutations, genes with hundreds of mutations but no hot spots and genes with hot-spot mutations. In this dissertation, we established the efficient and accurate DHPLC platform provide available testing procedures and protocols applying on several entities of diseases, such as alpha-thalassemia, beta-thalassemia, tuberous sclerosis complex, Duchenne/ Becker muscular dystrophy, and spinal muscular atrophy. Although many genetic diseases are caused by the presence of point mutations in respective genes, an increasing number of diseases are known to be caused by gene copy number changes. We want to develop a rapid and reliable method for quantitative analyses for human genome in various genetic disorders. The method involves amplifications of a test locus with unknown copy number and a reference locus with known copy number, following by DHPLC and CE techniques to detect single copy changes without the use of radioactive labeling. In this dissertation, we established the efficient and accurate gene dose determination system by using the DHPLC and CE platform based on multiplex PCR strategies and applying on several entities of diseases including Duchenne/ Becker muscular dystrophy, spinal muscular atrophy, Prader-willi syndrome, Charcot-Marie-Tooth disease and alpha-thalassemia. Moreover, once we have established this powerful system, we will further apply this technique on rapid detection of trisomy syndrome and microdeletion syndrome including trisomy 13, trisomy 18, DiGeorge syndrome, William’s syndrome, and others. Moreover, Polyalanine repeat expansions constitute a new group of repeat expansion disorders that differ from previously known expansion-associated diseases in many ways. They occur primarily in transcription factors with important roles during development. As a consequence, the clinical spectrum associated with these mutations consists mainly of congenital malformation syndromes. Based on the finding that protein misfolding and degradation are a major pathogenetic mechanism in polyalanine repeats, it is likely that the clinical phenotype is influenced by the expression of chaperones and other factors that affect this process. To explore the genetic regulating mechanism of polyalanine expansion and the pathophysiologic pathway, we conduct this pilot study. In the dissertation, we established the reliable comprehensive genetic testing for PHOX2B gene and for the other genes with abnormal polyalanine expansion. In summary, by using DHPLC, CE, melting curve analysis and MLPA techniques in genetic diagnosis, the advantages including that DHPLC got the powerful ability to identify random mutations, CE can the be adapted to gene dosage determination for high throughput screening in medical applications, melting curve analysis could identify the mutation site with highly efficient performance and MLPA allows detection of gene deletions, duplications, and rearrangements in whole gene. Compared to classic approaches of mutation screening, DHPLC, CE, melting curve analysis and MLPA could be valuable alternative in a more rapid, economic and highly sensitive way in genetic diagnosis.