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Copy number variation in human disease

Karina Meden Sørensen


With biological focus on heart diseases, the purpose of this project is to develop novel methods and to determine new biological markers for diagnostics of various diseases related to gene copy number variations (CNVs). One aim is to enable neonatal and/or prenatal screening as well as retrospective screening projects based on Guthrie cards, a valuable DNA source archived in a population based national biobank. Moreover, my aim was to develop a simple unifying screening platform for both known syndromes and newly recognised markers related to congenital heart diseases.
Current technologies for CNV detection in multiple loci require large amounts of DNA and are therefore not applicable for neonatal screening, where DNA sources are limited. Hence, method development is in focus within the area of DNA extraction and amplification. DNA extracted from neonatal filter paper blood spots using commercial kits often produce low yield and low concentration. At present time, this is insufficient for CNV analysis using common techniques. In order to overcome this problem three approaches were taken: 1) Whole genome amplification (WGA) of the DNA extract which produces large amounts of DNA, but has uncertainties regarding equal allele representation, 2) optimisation of the sensitivity of the techniques used for CNV analysis in order to be used on small amounts of DNA and 3) optimisation of the Guthrie card DNA extraction by development of a new in-house method. Four WGA systems were tested on DNA extracted from Guthrie cards to evaluate the amplification bias, concordance- and call rates, cost efficiency, and flexibility using a SNP detection system. All systems successfully amplified picograms of DNA to micrograms of product without loss of heterozygosity and with minimal allelic bias. Further evaluations using a modified protocol set up resulted in a concordance rate of 99.7% and a call rate of 99.8%. A real-time quantitative PCR method was evaluated regarding applicability in CNV determination and the Multiplex Ligation-dependant Probe Amplification (MLPA) technique was optimised in order to perform CNV detection on limited amounts of DNA. Cohorts of patients with schizophrenia, congenital adrenal hyperplasia (CAH), congenital heart disease (CHD) and DiGeorge syndrome were used as model systems.
Multiplex Q-PCR proved to be a robust and easy-to-perform technique for CNV analysis of a single locus, and MLPA was successfully optimised for CNV analysis on limited DNA material in multiple loci. In the attempt to determine new biological markers in CHD a MLPA probe mix was developed and successfully tested with DNA from children with known variations. A cohort of children with non-syndromic CHD not previously screened was subsequently analysed and several CNVs discovered. Copy number aberrations were detected in approximately 4% of the children. The extents of the chromosomal aberrations were established by array CGH and candidate genes were evaluated based on findings in the literature. Furthermore, analysis of non affected parents concluded that nearly 80% of the cases were familial, leaving us to conclude that the observed aberrations are not the stand-alone cause of heart malformation. The future perspectives of this heart disease kit are a more extensively screening of newborns with congenital heart diseases.