19 different combinations of mutations were detected: 3 isolates (2.2%) had four mutations, 8 (5.8%) had three mutations, 43 (31.2%) had double mutations and 16 (11.6%) had single mutations.
Only two of 15 (13.3%) cases with three gene mutations and eight of 27 (29.6%) cases with two gene mutations had a single subpopulation.
Three isolates (mutation profile type 2, 9 and 12) had four amino acid substitutions, 8 isolates (mutation profile type 4, 5 and 8) had three amino acid substitutions, 43 isolates (mutation profile type 1, 3, 6, 7, 10, 11 and 13) had double amino acid substitutions and 16 isolates (mutation profile type 14 19) had single amino acid substitutions.
In addition, in the C181S mutation, which has single palmitoyl and is Golgi targeted like N-Ras, more rasosomes are created than in H-Ras-WT or the singly palmytoylated C184S.
By contrast, the multiple mutations model [ 17- 19] assumes that beneficial mutations have a single characteristic selective value and can appear in any genetic background, including those containing beneficial mutations generated previously and still not fixed.
These analytical results demonstrated that the three single mutations had negative effects on the thermostability of AEXynM as predicted by total energy value calculation, among which one single mutation of C5T caused the most significant decrease in thermostability.
Although most of the single mutations had no effect, we cannot exclude the possibility that different phosphorylation sites may combine together to play a role in autophagy.
The other single mutations had no impact on affinity despite the fact that three of them were defined as hotspots and were strongly selected during the ribosome-display process.
However, we reasoned that if this mutation had a single ancestral origin, carriers would have more chromosomal segments in common than non-carrier patients.
However, despite the disruptive phenotypes observed in homotetrameric Thr441Val or Thr442Val channels, single subunit mutations had minimal effects in the background of a concatenated tetramer.
