Abstract:Purpose Description of the confluence of different molecular techniques to detect three different mutations in one cell. The man carries a 20 base pair insertion in exon 12 of the POR gene (c.1551_1552ins20), and the woman carries a point mutation in exon 8 of the POR gene (c.859G>C) plus a triplet repeat expansion in the HTT gene.
“…Fourteen single blastomeres were studied by PCR. IVF, embryo culture and biopsy procedures were performed according to previously described protocols [14].…”
Preimplantation genetic diagnosis represents a valid reproductive option for couples affected of propionic acidemia, in order to avoid transmission to offspring.
“…Fourteen single blastomeres were studied by PCR. IVF, embryo culture and biopsy procedures were performed according to previously described protocols [14].…”
Preimplantation genetic diagnosis represents a valid reproductive option for couples affected of propionic acidemia, in order to avoid transmission to offspring.
“…Out of the 13 panel markers, 6 have been previously reported and used successfully in PGD (11)(12)(13)(14)(15)19 ). The remaining 7 markers (HD2098407, HD2362117, HD2417179, HD3139793, HD3377975, HD3615631, and HD3829173), each possessing a PIC value Ͼ0.5, are novel.…”
BACKGROUND
Preimplantation genetic diagnosis (PGD) of Huntington disease (HD) generally employs linkage analysis of flanking microsatellite markers to complement direct mutation testing, as well as for exclusion testing. Thus far, only 10 linked markers have been developed for use in HD PGD, with a maximum of 3 markers coamplified successfully. We aimed to develop a single-tube multiplex PCR panel of highly polymorphic markers to simplify HD PGD.
METHODS
An in silico search was performed to identify all markers within 1 Mb flanking the huntingtin (HTT) gene. Selected markers were optimized in a single-tube PCR panel, and their polymorphism indices were determined in 2 populations. The panel was tested on 63 single cells to validate its utility in PGD.
RESULTS
We identified 102 markers in silico, of which 56 satisfied the selection criteria. After initial testing, 12 markers with potentially high heterozygosity were optimized into a single-tube PCR panel together with a 13th more distally located marker. Analysis of DNA from 183 Chinese and Caucasian individuals revealed high polymorphism indices for all markers (polymorphism information content >0.5), with observed heterozygosities ranging from 0.5–0.92. All individuals were heterozygous for at least 5 markers, with 99.5% of individuals heterozygous for at least 2 markers upstream and downstream of the HTT CAG repeat.
CONCLUSIONS
The tridecaplex marker assay amplified reliably from single cells either directly or after whole genome amplification, thus validating its standalone use in HD exclusion PGD or as a complement to HTT CAG repeat expansion-mutation detection.
“…After thawing, 23 zygotes survived and 15 cleaving embryos were available for biopsy on day +3. Biopsy procedures were performed according to previously described protocols [1].…”
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