Rapid genotyping of common MeCP2 mutations with an electronic DNA microchip using serial differential hybridization

Abstract

Rett syndrome is a neurodevelopmental disorder that affects females almost exclusively, and in which eight common point mutations on the X-linked MeCP2 gene are knows to cause over 70% of mutation-positive cases. We explored the use of a novel platform to detect the eight common mutations in Rett syndrome patients to expedite and simplify the process of identification of known genotypes. The Nanogen workstation consists of a two-color assay based on electric hybridization and thermal discrimination, all performed on an electronically active NanoChip. This genotyping platform was tested on 362 samples of a pre-determined genotype, which had been previously identified by a combination of DHPLC (denaturing high performance liquid chromatography) and direct sequencing. This genotyping technique proved to be rapid, facile, and displayed a specificity of 100% with 3% ambiguity. In addition, we present consecutive testing of seven mutations on a single pad of the NanoChip. This was accomplished by tagging down two amplimers together and serially hybridizing for seven different loci, allowing us to genotype samples for seven of the eight common Rett mutations on a single pad. This novel method displayed the same level of specificity and accuracy as the single amplimer reactions, and proved to be faster and more economical.

Figure 1.

Schematic diagram of the Nanogen differential hybridization process. A: Attachment of a single set of PCR amplimers on one pad of the NanoChip. B: Fluorescent oligonucleotide probes specific to the wild-type (labeled with Cy-3) and mutant (labeled with Cy-5) base are added to the PCR product-loaded pad. In this case, one probe is a match, the other is mismatched for the base in question. In addition, a 27 to 33-mer stabilizer oligo is added to the pad simultaneously to bind to the complementary area directly 5′ of the mutation. C: Thermal stringency then strips an improperly binding probe while leaving the correct one bound. D: Multiplex binding of two amplimers to the same pad of the NanoChip. The subsequent probing is done serially with distinct probes and stabilizers for one amplimer, followed by thermal stringency and washing, and then the next amplimer is tested.

Figure 2.

Nanogen Reader results for four samples that were heterozygous for MeCP2 mutations and six samples that were homozygous wild-type. After loading onto NanoChips, samples were read by the Nanogen Reader. Arbitrary fluorescence units were measured for both wild-type (Cy-3) and mutant (Cy-5) signals in all samples.

Figure 3.

Nanogen system results for 368 tested samples. After detection of fluorescence, wild-type (Cy-3) and mutant (Cy-5) signals were normalized by a control heterozygous sample. The Nanogen software then used the ratio of the normalized data, shown above, to genotype the sample. The generated results were in agreement with those from previous DHPLC and sequencing.

Figure 4.

Nanogen system results for testing of seven samples. For the samples, each point mutation was probed serially. After detection of fluorescence, calls were made in the same manner as non-multiplexed samples and probes were subsequently stripped, readying the samples for the next test. The generated results were in agreement with those from previous DHPLC and sequencing.