Development of adnvanced technology for the detection of mutations and polymorphisms in DNA and RNA

Doctoral Dissertation uoadl:1305662 179 Read counter

Τομέας Υγείας - Μητέρας - Παιδιού
Library of the School of Health Sciences
Deposit date:
Πούλου Μυρτώ
Dissertation committee:
Εμμανουήλ Καναβάκης, Αριάδνη Καλπίνη-Μαύρου, Κωνσταντίνος Τρούγκος
Original Title:
Ανάπτυξη προηγμένης τεχνολογίας για την ανίχνευση μεταλλάξεων και πολυμορφισμών σε DNA και RNA
Translated title:
Development of adnvanced technology for the detection of mutations and polymorphisms in DNA and RNA
The progress in genome research as well as the increasing data coming from
genome wide analysis programs, dictate the need for development of novel
methodologies for molecular analysis of the genes involved in the occurrence of
disease. The aim of this PhD thesis was the design and standardization of high
throughput technology for the detection of DNA variants. Considering the quick
turnaround time of a diagnostic laboratory, another aim was the establishment
of a reliable algorithm by which all novel variants could be assessed
concerning their effect on the protein produced.
Using as disease models two autosomal recessive genetic disorders (Cystic
Fibrosis – CFTR gene and Wilson disease – ATP7B) we designed three different
protocols for the platforms NanoChip400TM, Luminex xMAPTM and LightScanner. The
principle of function for the platform ΝanoChip400TM is based on the electronic
operation of an electrical field and therefore is an electronic microarray
system. The design was for ATP7B gene. The principle of function for the
platform Luminex xMAPTM is flow cytometry and therefore is a system that
combines advanced fluidics, optics, and digital signal processing with
microsphere technology. The design was for CFTR and ATP7B genes. The principle
of function for the LightScanner platform is the DNA melting due to heating and
therefore is a system able to perform High Resolution Melting Analysis. The
design was for CFTR and ATP7B genes.
Using the NanoChip400TM we analysed 144 neonatal samples from the island of
Kalymnos and Crete. We detected 14 healthy heterozygotes for the mutation
p.His1069Gln (6 from Kalymnos and 8 from Crete), 2 healthy heterozygotes for
the mutation p.Arg969Gln (from Kalymnos) and 2 asymptomatic patients with
genotype p.[His1069Gln] + [His1069Gln] (from Kalymnos)
Using the Luminex xMAPΤΜ we tried to detect έγινε the 50 most common mutations
of the CFTR gene in the Greek population. Despite all the modifications of the
test protocol, we failed to obtain the adequate signals for all the mutations.
For the ATP7B gene, we managed to get acceptable values for all the mutations
and to genotype correctly 100 samples for 26 mutations.
Using LightScanner, during standarization we tested sucesfully 100 known
samples and 100 blind samples for each disease. The sensitivity and specificity
was calculated at 100%. Another very important result was the standardzation of
a protocol for prenatal diagnosis and for preimplantation genetic diagnosis.
A reference laboratory for a genetic disease should be able to offer the
maximum possible mutation detection rate. This assumption takes a special
meaning when we are referring to a genetic disorder with a high heterogeneity
rate in the test population. Based on the above, the most suitable method out
of the three different ones developed during this PhD thesis is High Resolution
Melting analysis. This method offers high accuracy and sensitivity, can be
applied without the need for prior characterization of the test population and
can readily detect any genetic variant allowing for maximum detection rate.
Cystic Fibrosis, Wilson Disease , High Resolution Melting Analysis, Prenatal Diagnosis, Preimplantation Genetic Diagnosis
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