Figure 1
Flowchart Illustrating the Strategy and Results of this Study. The scheme summarized the different experimental steps and main results obtained.
Table 1
Comparison of the Frequencies of Rare Variants in Six Selected Genes in TS and Control Populations
| Gene | Type of variants | TS cohort (120 individuals) | gnomAD (138,632 individuals) | p | Control individuals (788 individuals) | p |
|---|---|---|---|---|---|---|
| OPRK1* | Missense/LoF (MAF<1%) | 5 (4.2%) | 1099 (0.8%) | 0.003 | 4 (0.5%) | 0.003 |
| Damaging/LoF (MAF<1%) | 4 (3.3%) | 541 (0.4%) | 0.001 | 3 (0.4%) | 0.007 | |
| OPRM1** | Missense/LoF (MAF<1%) | 8 (6.7%) | 8,085 (5.8%) | 0.694 | 22 (2.3%) | 0.048 |
| Damaging/LoF (MAF<1%) | 5 (4.2%) | 3,786 (2.7%) | 0.268 | 10 (1.2%) | 0.037 | |
| NTS | Missense/LoF (MAF<1%) | 3 (2.5%) | 998 (0.7%) | 0.057 | 10 (1.2%) | 0.397 |
| Damaging/LoF (MAF<1%) | 2 (1.7%) | 474 (0.3%) | 0.064 | 2 (0.25%) | 0.087 | |
| NTSR1 | Missense/LoF (MAF<1%) | 12 (10.0%) | 14,333 (10.3%) | 1.000 | ND | |
| Damaging/LoF (MAF<1%) | 6 (5.0%) | 8,248 (5.9%) | 0.847 | ND | ||
| NTSR2 | Missense/LoF (MAF<1%) | 4 (3.3%) | 3,142 (2.3%) | 0.353 | ND | |
| Damaging/LoF (MAF<1%) | 4 (3.3%) | 1,772 (1.3%) | 0.069 | ND | ||
| PCDH10 | Missense/LoF (MAF<1%) | 4 (3.3%) | 2,494 (1.8%) | 0.171 | ND | |
| Damaging/LoF (MAF<1%) | 3 (2.5%) | 1,174 (0.8%) | 0.083 | ND |
The details of variants included in the comparisons appear in supplementary Tables 10 and 11. The number of variants/individuals calculated from gnomAD (supplementary Table 11) assumed that rare variants correspond to one individual each. Variant frequencies were compared with Fisher’s exact tests.
Figure 2
Knockdown of the Zebrafish OPRK1 Orthologue Results in Hyperactivity during Development. (A) Activity heatmap illustrating spontaneous movement of 28 hpf embryos inside the chorion during a 10 seconds period. Higher levels of activity are evident in embryos injected with oprk1 splice-blocking AMO (spOPRK1) or ATG-targeting AMO (OPRK1 KD) when compared to mismatch AMO-injected embryos (Mismatch control). Expression of human OPRK1 transcript (hOPRK1) rescues the hyperactivity phenotype. (B) RT-PCR product of amplification of a region spanning exons 2 and 3 of the zebrafish oprk1 transcript. Injection of the splice-blocking AMO (spOPRK1) results in a defective splicing in this region. (C) Quantification of the total activity of 28 hpf zebrafish embryos. (D) Quantification of the frequency of movement bursts in the 28 hpf embryos.