MELAS mutant load was quantified using a semiquantitative fluorescent PCR- HaeIII restriction test.
The m.3243A > G mutant load was assessed in 31 whole embryos and 70 single blastomeres (Table 3).
In the 15 remaining embryos, assessment of the mutant load was performed on the embryo in toto.
The mutant load was calculated by dividing the mutant peak area (73 bp) by the sum of normal (171 bp) and mutant (73 bp) peak areas.
The m.3243A > G mutant load was found to be highly heterogeneous among various embryos from a given individual, ranging from 18 to 50% and from 0 to 77% in patients' 1 and 2 embryos, respectively.
When pooling values from all cells analyzed for a given individual, average mutant load was very close to that achieved from the overall lymphocyte extract recovered from a 10-ml blood sample.
When placenta and other tissues were available at the same term of gestation (three fetuses), values were similar in two fetuses (74.6 vs. 74% and 78 vs. 78% for fetus 2a and 7a, respectively), while fetus' 2b mutant load was higher in placenta than in other tissues (57 ± 2% vs. 42 ± 2%, respectively).
Mutant loads were assessed in extraembryonic or embryonic tissues collected between 10 GW to term, in 12 fetuses from 7 carriers (families 2 to 8; Table 4).
When pooling values from all cells analyzed for a given cell type in a given fetus, the average mutant loads were very close to those achieved from overall chorionic villi sampling (CVS) and amniotic fluid sample (AFS) mutant loads (variation <10%; Fig. 2).
When excluding data from extra-embryonic tissues (10 GW trophoblast or at term placenta), m.3243A > G mutant loads were identical in all tested tissues from a given fetus (mean ± SD: 74.6 ± 0.7% [three tissues], 42 ± 0.8% [five tissues], 71±2%2% [five tissues], and 78 ± 0.9% [seven tissues] for fetuses 2a, 2b, 5b and 7a, respectively).
