PMC8578848_01
(A) Detection of MECP2 exon alterations via multiplex ligation-dependent probe amplification (MLPA). The arrows illustrate the presumed deletions, as the area of the peaks from the female proband DNA is approximately half that of the female controls. (B) The MECP2 amplicon with MECP2-real time quantitative PCR-2 primers to narrow down the deletion breakpoints and determine the results of MLPA. MECP2 relative amount of female proband, and male fetus were half that of female control. (C) The products of long-range PCR were analyzed with 1.5% agarose gel electrophoresis. The PCR product from the proband showed three DNA bands, whereas PCR product bands of the proband's parents and the fetus, were single. (D) Three DNA bands of long-range PCR from the proband were separately cut, T-A cloned, and finally sequenced after plasmid extraction. The sequence of top DNA band was wild type; both, bottom and middle DNA bands show 713-base pairs deletion in exon 4 (c.441_1153del713), and the single band of proband's parents and the fetus were of the wild type. (E) There was no difference between the PCR products untreated (left) and treated with heating and reannealing (right) in electrophoresis analysis. The treated PCR products were denatured at 95  C for 3 min, followed by gradual reannealing for 30 min with a temperature ramp of -1  C/min to optimize for the formation of heteroduplexes and homoduplexes. (F) Schematic diagram of three DNA bands after PCR. The top DNA band was homoduplex of wild type, the bottom DNA band was homoduplex of mutant type, the middle DNA band was heteroduplex of wild type and mutant type. Part B (red): 713-base pairs deletion in exon 4 (c.441_1153del713) of MECP2.
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Additionally, a heterozygous deletion of exon 4 in MECP2 was found in the proband (Figure 1A). We then used three pairs of real-time qPCR primers to narrow down and identify the deletion breakpoints (see Supplemental Information, Supplementary Figures 1A,B, Figure 1B). The product of long-range PCR (upon running a gel electrophoresis) showed three DNA bands of the proband (Figure 1C). Surprisingly, not only the bottom band, but also the middle one, showed a 713-base pair deletion in exon 4 of MECP2 (c.441_1153del713) (Figure 1D). As shown in Figure 1F, for base pairing, the mutated chain combined with the wild chain around the common parts (parts A and C), but the unpaired part (part B) of the wild chain may form a particular structure. As shown in Figure 1E, by the classical method of producing heteroduplexes, untreated PCR products (left) and those treated with heating and re-annealing were the same, further proving that the middle band was heteroduplex (Figure 1E).