Identification and characterization of NOS1 mutations in CHH probands (a) Lollipop plot illustrating the distribution of identified mutations in functional domains (blue boxes) of the human NOS1 protein (upper panel) and in highly constrained subregions (LIMBR score < 5%; lower panel in red). (b) Pedigrees of CHH probands harboring NOS1 mutations. Phenotypes are indicated by symbols as shown in the legend (bottom). (c) Representative western blot showing ectopic expression of NOS1 protein (Anti-Myc tag) in HEK293 cells 48h after transfection with WT or mutant NOS1 constructs.

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Context 1

... is the most commonly occurring isoform in the nervous system (19). Through exome sequencing of a large cohort of unrelated subjects with CHH (n = 341), we identified five ultra-rare heterozygous NOS1 missense variants in six probands (~2%) ( Table 1; Table S1; Figure 2). None of the probands harbored pathogenic or likely pathogenic variants in known CHH genes, according to the ACMG classification. ...

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... identified NOS1 mutants mapped to highly constrained sub-regions of NOS1 critical for protein function (Figure 2a,b). Three variants (p.Thr1107Met, p.Glu1124Lys and p.Ile1223Met) were located in the C-terminal reductase domain, critical for the catalytic activity of the protein. ...

Context 3

... specifically, p.Ile1223Met was located in the NAD-binding pocket and the p.Thr1107Met and p.Glu1124Lys in the FAD-binding pocket, both essential for electron transfer to the oxygenase domain of the adjacent subunit of the dimer, leading to NO formation (22). The p.Ala231Thr mutation lay within a regulatory region, the protein inhibiting NOS1 (PIN)-binding domain, while p. Arg260Gln was located in a lowcomplexity region between the PIN-binding and oxygenase domains (Figure 2a). ...

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... DNA was available in four cases. All probands inherited their mutations from unaffected or partially affected parents (Figure 2b). In Family D, the female proband (II-1) harboring a p.Glu1124Lys NOS1 mutation exhibited KS and inherited the NOS1 mutation from her father (I-1) with constitutional delay of growth and puberty (CDGP), a transient form of GnRH deficiency (23). ...

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... testing the enzymatic activity of the NOS1 mutants identified above in vitro, we first assessed their ectopic expression after transient transfection of HEK293 cells with tagged wild-type (WT) and mutant NOS1 cDNA. Western-blot analysis revealed that, in contrast to the WT construct, Thr1107Met and Glu1124Lys mutants were barely detectable, suggesting disrupted protein synthesis or rapid degradation (Figure 2c). Consistent with altered expression, calcium-induced NO release using live-cell imaging was abrogated in cells transiently expressing Thr1107Met and Glu1124Lys mutants, and significantly attenuated for the 3 other reported mutants compared to cells expressing the WT plasmid (p < 0.001; Figure 2d-f; Figure S2a,b), suggesting decreased NOS1 activity. ...

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... analysis revealed that, in contrast to the WT construct, Thr1107Met and Glu1124Lys mutants were barely detectable, suggesting disrupted protein synthesis or rapid degradation (Figure 2c). Consistent with altered expression, calcium-induced NO release using live-cell imaging was abrogated in cells transiently expressing Thr1107Met and Glu1124Lys mutants, and significantly attenuated for the 3 other reported mutants compared to cells expressing the WT plasmid (p < 0.001; Figure 2d-f; Figure S2a,b), suggesting decreased NOS1 activity. NOS1 requires homodimerization to enzymatically convert L-arginine and oxygen into L-citrulline and NO (24), and NOS1 mutants can impair the formation of active NOS1 dimers, resulting in reduced NO production in vitro (25). ...

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... requires homodimerization to enzymatically convert L-arginine and oxygen into L-citrulline and NO (24), and NOS1 mutants can impair the formation of active NOS1 dimers, resulting in reduced NO production in vitro (25). The decreased enzymatic activity of mutants was further confirmed using a fluorometric nitrate kit ( Figure S2c). To test the possibility that NOS1 mutants impair the activity of NOS1 dimers by heterodimerizing with the NOS1 produced by the WT allele, we generated bicistronic constructs producing equimolar amounts of WT and mutated NOS1 transcripts ( Figure S2d). ...

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... decreased enzymatic activity of mutants was further confirmed using a fluorometric nitrate kit ( Figure S2c). To test the possibility that NOS1 mutants impair the activity of NOS1 dimers by heterodimerizing with the NOS1 produced by the WT allele, we generated bicistronic constructs producing equimolar amounts of WT and mutated NOS1 transcripts ( Figure S2d). NOS1 activity in vitro was diminished to the same extent by the biscistronic construct as when cells were transfected with the mutants alone ( Figure S2c), and mutant isoforms were seen to co-immunoprecipitate with WT isoforms (Figure 2g), demonstrating that the NOS1 mutants identified in our patients are dominant negative. ...

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... test the possibility that NOS1 mutants impair the activity of NOS1 dimers by heterodimerizing with the NOS1 produced by the WT allele, we generated bicistronic constructs producing equimolar amounts of WT and mutated NOS1 transcripts ( Figure S2d). NOS1 activity in vitro was diminished to the same extent by the biscistronic construct as when cells were transfected with the mutants alone ( Figure S2c), and mutant isoforms were seen to co-immunoprecipitate with WT isoforms (Figure 2g), demonstrating that the NOS1 mutants identified in our patients are dominant negative. ...

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... test the possibility that NOS1 mutants impair the activity of NOS1 dimers by heterodimerizing with the NOS1 produced by the WT allele, we generated bicistronic constructs producing equimolar amounts of WT and mutated NOS1 transcripts ( Figure S2d). NOS1 activity in vitro was diminished to the same extent by the biscistronic construct as when cells were transfected with the mutants alone ( Figure S2c), and mutant isoforms were seen to co-immunoprecipitate with WT isoforms (Figure 2g), demonstrating that the NOS1 mutants identified in our patients are dominant negative. ...