The sequencing of the human genome, coupled with advances in genomics technologies, is having a major impact on understanding basic human biology, as well as the molecular causes of diseases and toxicities . In fact, the leaders of the U.S. Food and Drug Administration (FDA) and the National Institutes of Health (NIH) have recently outlined the opportunities in clinical medicine that are being created by advances in basic science, including genomics . The two agencies have announced a new collaborative effort to focus regulatory and translational sciences on bringing medical products and therapies into the age of personalized medicine. This growing store of knowledge has enormous potential application to the development of devices and tests for use in the diagnosis, mitigation, treatment, cure, and prevention of disease and other conditions.
As of 2010, more than 100,000 germline mutations in more than 3700 genes have been associated with human inherited disease, with about 300 new disease genes and 10,000 mutations being identified annually . In principle, DNA tests could be developed for each of these conditions. Development and approval of such tests for clinical diagnostics often require that accuracy be demonstrated on patient samples. That is, the test needs to detect the disease-causing mutation with high precision and accuracy. In addition, measures of proficiency are required by testing laboratories to ensure continued accuracy of the results. The Clinical Laboratory Improvement Advisory Committee has recently issued a good practices report for molecular genetic testing and has stressed the need for performance assessments . Such recommendations are supportive of the 2008 Report of the Secretary’s Advisory Committee on Genetics, Health, and Society (SACGHS) . Additionally, the SACGHS has identified the development of genetic reference materials as one of five critical gaps in the oversight system of genetic testing . The device approval process, use of reference and control materials, and ongoing proficiency assessments require relatively large quantities of clinical samples in order to assure test performance is adequate and maintained over time. The availability of sufficient samples with appropriate mutations has been recognized as a critical issue in genetic testing, given the paucity of validated clinical inherited disease gene samples . Therefore, methods that can expand the limited supply of validated clinical samples would have major impacts on both the test and device performance evaluation and approval process, and the on-going proficiency assessment of the test providers.
The importance of the amount of template DNA for MDA-based WGA has been thoroughly discussed for SNP assays and STR genotyping in previous studies [28, 31]. For example, large number of SNPs could be accurately detected from as low as 0.01 ng of DNA template [27, 28] and even degraded DNA samples can be used for forensic SNP typing , while over 100 ng of DNA template was needed for optimal STR genotyping . In this study, we mainly focused on evaluating the uniformity and fidelity of WGA DNAs using CGH arrays, TaqMan copy number assays, and DNA sequencing. The results indicated the amplified DNA and its native unamplified DNA that we examined were similar, although not identical, in terms of DNA copy number variation. Importantly, no detectable introduced mutations were found under our experimental conditions.
The Agilent Human CGH array used in this study includes approximately 970,000 probes at 1 to 2 kb intervals throughout the human genome. To ensure the quality of this study, four independent replications for both the WGA and aCGH processes were used. The self-self hybridization of unamplified Normal DNA samples provided us the base-line measurement of noise for aCGH technology. The box plot of unamplified Normal samples indicated less than 0.004% of the approximately 970,000 probes on the CGH array have fold changes greater than 1.5 and no CNVs can be detected by aCGH (Figure 2 and Table 3). The low Kolmogorov distances (0.025) and high Phi correlations (1.00) between the four replicates of unamplified samples further showed the aCGH technology was reproducible and could be a reliable tool to access variability of the WGA process.
The WGA technologies have evolved over the years from Taq DNA polymerase-based (PCR-based) to bacteriophage Ф29 DNA polymerase-based methods (MDA). We focused on MDA methods in this study because of the processivity and reportedly low replication error rate of the Ф29 DNA polymerase. In this study, we compared two MDA-based commercial WGA kits: the REPLI-g and GenomiPhi kits. The comparison between the unamplified and amplified DNA samples showed that relatively few probes (approximately 1% or less) were over- or under-amplified by more than 2-fold (Table 3). GenomiPhi-amplified DNA, however, showed consistently more CNVs than the REPLI-g-amplified DNA. Kolmogorov distances also indicated that differences in the distribution of DNA copy number existed between the unamplified and amplified samples (Table 2), and that these differences were greater than the differences between the REPLI-g and GenomiPhi amplified samples (Table 7). Thus, even though the kits differ in method of DNA denaturation (heat vs. alkaline), buffer composition, and reaction time (4 hr vs. 10 hr), they produce similar amplified DNA, likely the result of the common Ф29 DNA polymerase.
The data in this report indicate that the two WGA methods examined can consistently amplify small amounts of DNA (ng) to large quantities (~40 μg) with relatively small changes in DNA copy number along the chromosomes. Changes in DNA copy number of greater than 3-fold are evident as under-amplified regions at the ends of chromosomes, as illustrated in Figure 3, and in discrete regions on many of the chromosomes (Table 4). Because of the consistency of the DNA amplification, knowledge of the positions of likely under-amplification can prevent the inappropriate use of WGA DNA. Results presented in Table 4 may serve as a reference guide to avoid target genes in these regions. Inspection of the GC content within these consistently under-amplified regions showed an average GC content of nearly 53% which was higher than the overall GC content of the human genome of 41%. Further investigations will be needed to evaluate the significance of this observation.
Cystic fibrosis (CF) is caused by severe dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR), which commonly leads to progressive lung disease and a shortened life . Currently, there is no cure available for CF, even though multiple interventions have been developed to slow its progression. Since the FDA cleared the first genotyping assay for CF in 2005, manufacturers have developed a number of genotyping tests for rare heritable diseases caused by multiple mutant alleles, using patient samples to assess performance. There is an increased interest in using WGA DNA samples created from patient samples to support the performance of the assays. This study shows that within certain limits, the WGA process produces large quantities of DNA that may be useful for this purpose. To address the concerns of mutations introduced by WGA, TaqMan copy number assays were used to analyze CNVs at 10 locations within CFTR gene region and a random selected 2 kb region in CFTR gene was also sequenced. Results from the TaqMan copy number assay are in excellent agreement with the aCGH results. The DNA sequencing analysis showed there were no mutations induced by WGA in this approximately 2 kb region of the CFTR gene (mutation induction was less than 4 x 10-5), which indicates that WGA DNAs can be used for enrichment of DNA samples for cystic fibrosis genotyping assays.
The objective of the study presented here was to determine whether WGA amplified samples may be a reliable alternative to native clinical specimens for assessing the performance of a test under investigation. The conclusions from this study provide scientific input that may serve to support regulatory decisions in the ascertainment of safety and effectiveness of diagnostic products that use whole genome amplified samples in clinical studies. This study may serve as a guide to the technical qualification of WGA DNA for assessing the performance of genotyping assays.