Table 2.
Evaluation of functional studies examining MSH6 variants.
| Assay | MMR Activity | 6-TG Selection | Nuclear Localization | Mechanistic | Splicing | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Reference | [44] | [43] | [42] | [40] | [48] | [41] | [47] | [50] | [51] | [53] | [54] | [23] | [64] | [65] | [47] | [51,71,74,75,76,77,78,79,80,81,82,83,84,85,86] |
| Material used | Human cDNA in baculovirus vector | Human cDNA in baculovirus vector | Human cDNA in baculovirus vector | Human cDNA in baculovirus vector | Human cDNA in pCITE4a | Human cDNA in baculovirus vector | Human cDNA in baculovirus vector | Human cDNA in pCITE4a | Human cDNA in pCITE4a | Mouse DNA | Mouse DNA | Human cDNA in EGFP-C1 | Human cDNA in pEGFP-N1 | Human cDNA in baculovirus vector | Human cDNA in baculovirus vector | Human mRNA or minigene DNA constructs |
| Expression system | Sf9 insect cells | Sf9 insect cells | Sf9 insect cells | Sf9 insect cells | IVTT | Sf9 insect cells | Sf9 insect cells | IVTT | IVTT | mESC | mESC | DLD-1 cells | HEK293 cells | Sf9 insect cells | Sf9 insect cells | RT-PCR |
| Functional output | Mismatch repair activity | Mismatch repair activity | Mismatch repair activity | Mismatch repair activity | Mismatch repair activity | Mismatch repair activity | Mismatch repair activity | Mismatch repair activity | Mismatch repair activity | Sequencing of 6TG resistant colonies | Sequencing of 6TG resistant colonies | Nuclear/cytoplasmic localization | Nuclear/cytoplasmic localization | Multiple outputs | Multiple outputs | cDNA sequence |
| Wild-type control | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes | - | yes | yes | Yes | Yes | Yes | - |
| Positive control | Yes (HCT- 15 cells) | Yes (HCT- 15 cells) | Yes (HCT- 15 cells) | Yes (LoVo cells) | 2 pathogenic variants | Yes (LoVo cells) | Yes (LoVo cells) | 52 pathogenic variants | 1 pathogenic variant | 1 pathogenic variant | 5 pathogenic variants | - | - | - | - | - |
| Negative control | Yes (TK6 cells) | Yes (TK6 cells) | Yes (TK6 cells) | Yes (HeLa cells) | 3 benign variants | Yes (HeLa cells) | Yes (HeLa cells) | 31 benign variants | - | - | 4 benign variants | - | - | - | - | - |
| No. of VUS examined | 4 | 2 | 5 | 1 | 15 | 6 | 1 | 7 | 10 | 3 | 26 | 4 | 10 | 7 | 1 | - |
| Technical replicates | 2 | 2 | 2 | 3 | 3-4 for VUS and >6 for controls | 3 | - | >3 | 3-4 | - | - | - | 3 | 3 | 3 | - |
| Biological replicates | - | - | - | - | - | - | - | - | - | yes | yes | - | - | - | - | - |
| PS3/BS3 criterion [87] | Max PS3_Supporting/Max BS3_Supporting | Max PS3_Supporting/Max BS3_Supporting | Max PS3_Supporting/Max BS3_Supporting | Max PS3_Supporting/Max BS3_Supporting | Max PS3_Supporting/Max BS3_Supporting | Max PS3_Supporting/Max BS3_Supporting | Max PS3_Supporting/Max BS3_Supporting | Max PS3_Very_Strong/Max BS3 | Max PS3_Very_Strong/Max BS3 | Do no use PS3/BS3 | Max PS3_Supporting/Max BS3_Supporting | Do no use PS3/BS3 | Do no use PS3/BS3 | Do no use PS3/BS3 | Do no use PS3/BS3 | Max PS3_Very_Strong/Max BS3 |