High-throughput detection of submicroscopic deletions and methylation status at 15q11-q13 by a photo-cross-linking oligonucleotide hybridization assay

Abstract

Background: Current technologies for assessing genetic deletions and duplications of greater than one kilobase are labor-intensive or rely on PCR-based methods, and none offers the ability to simultaneously detect dosage abnormalities, assess 5′-to-3′ cytosine-guanosine (CpG) methylation, and interrogate single-nucleotide polymorphisms (SNPs). We describe a high-throughput platform for direct gene-dosage determination capable of concurrent assessment of other forms of gene modification. Methods: We used a light-activated interstrand nucleic acid cross-linking system (XLnt™ technology) to determine gene dosage at the 15q11-q13 deletion/duplication locus. We incorporated restriction enzyme digestion of genomic DNA into the method to assess CpG methylation in parallel with gene dosage. For method validation we used DNA from 31 cell lines with previously characterized 15q11-q13 gene dosage and parental origin status. Diagnostic cutoffs were set at 0.5 ± 0.15, 1 ± 0.15-0.25, and 2 ± 0.3. Results: Dosage-only experiments showed discrimination of deletions (n = 21) from healthy controls (NCs; n = 27) in all samples. Five of 49 samples gave results outside of specification. Concurrent evaluation of dosage and CpG methylation yielded dosage results within specification for 18 of 19 deletion and 8 of 12 NC samples. Paternal deletion and NC methylation pattern results were within specification in 17 of 19 and 9 of 12 runs, respectively. No overlap was demonstrated between value sets for the two groups. Conclusions: The XLnt technology provides a rapid, high-throughput platform for the accurate determination of gene dosage. The flexibility of this technology allows parallel interrogation of gene dosage, CpG methylation, and SNPs. © 2002 American Association for Clinical Chemistry.