how can I improve the following discussion"To the best of our knowledge, these are the first Iranian cases with IHPRF1 syndrome to be reported. We identified three novel homozygous variants in the NALCN gene that cause IHPRF1 syndrome in the probands. Our findings show, compared to the missense variant, the homozygous truncating variants (a stop gain and a splice site mutation) lead to more severe symptoms, especially with regard to the motor and cognitive development in probands. Additionally, the proband with the missense mutation could also walk, whereas the other two probands with the truncating variations could never even sit independently. This is in line with previous cases of IHPRF1, in which patients had more severe phenotypes due to splice site, nonsense, or frameshift mutations 17. This could be because missense mutations still allow the NALCN protein to function. The location of the mutation and whether it occurs in critical domains or protein segments, however, also affects how the mutation will behave. Figures 4a and 4b display the locations of the three novel homozygous variants. Two of these mutations probably cause nonsense-mediated decay (NMD), leading to the elimination of wild-type protein expression in the affected probands18. Third mutation happened in the crucial region that is important NALCN channelosome function19,20. Since the pathogenic c.1434+1G>A variation in Case 1 disrupts a gene's intron 12's highly conserved splice donor location in the center of the gene, it most likely causes the protein to have an NMD. (Figure 4a). Figure 4. NALCN gene and protein structure. a) The gene structure of NALCN is depicted and the position of splice site mutation is shown by red arrow. b) Transmembrane topology of NALCN protein and the localization of missense and nonsense mutation is shown. The transmembrane topology structure of SLC13A5 was adopted from Kang and Chen. c) The cryo-EM map of NALCN channelosome consists of NALCN-FAM155A-UNC79-UNC80 complex. The pathogenic c.3269G>A; p.W1090* variant thus result in a premature stop codon at position 1090 of the protein in case 2 (Figure 4b). It could also affect splicing site, which would interfere with the splicing process. Tryptophan 1090 is located in domain III of the NALCN protein. Even if the stop gain mutation (c.3269G>A; p.W1090*) does not cause the NMD of the protein, a stop gain mutation in the midst of domain III will surely affect NALCN function. This is so because domain IV of the NALCN protein is essential for building the pore shape, selectivity filter, and activation mechanisms. It is worth mentioning that Tryptophan amino acid at position 1090 is located at the extracellular loops III (ECLIII) segment, which interacts with L364 of FAM155A protein which is located in a loop that connects alpha helices in the C-lobe of FAM155A. As mentioned earlier, FAM155A is one of the proteins interacting with NALCN to form the NALCN channelosome along with UNC79, and UNC80 (Figure 4c). FAM155A protein forms extensive interactions with ECL of NALCN and possibly contributes to NALCN's membrane stabilization. In addition, FAM155A interacts with several additional amino acids in the ECLIII and ECLIV portions following W1090. The interaction between NALCN and FAM155A4 depends on these amino acids 4. Therefore, a stop gain mutation in the 1090 position not only disrupts NALCN function but also disrupts NALCN interaction with FAM155A. Therefore, even if the truncated protein escapes from NMD to some degree, it would not be able to assemble into a functional protein and subsequently a functioning channelosome. The c.2648G>T; p.G883V variant, on the other hand, is a missense mutation that leads to the substitution of glycine with valine at the position 883 (Figure 4b). This amino acid is found in the domain III segment S1Na of the NALCN protein. Glycine amino acid is unique among all amino acids because its side chain only consists of a hydrogen molecule. This property gives glycine greater conformational flexibility than other amino acids, which allows it to offer flexibility for other adjacent amino acids. As is shown in Figure 4b, segment S0 of domain III is an intracellular part of the protein that connects the DII-DIII linker to the S1 segment of domain III. DII-DIII linker is crucial in the interaction of NALCN with UNC79-UNC80 heterodimer for forming NALCN channelosome. UNC-interacting motifs (UNIM-B, C) that reside on the DII-DIII linker interact with UNC79-UNC80 heterodimer. The C-terminal domain (CTD) interacting helix (CIH) is another part located on the DII-DIII linker adjacent to domain III. CIH interacts with CTD and acts as an autoinhibitory mechanism for the NALCN channelosome. The binding of UNC79-UNC80 heterodimer to UNIM-C leads to the dissociation of CIH and CTD, releasing the auto-inhibition of NALCN by CIH and hence activating the NALCN channelosome. The glycine at the position 883 could be vital since it could provide flexibility and act as a hinge in the interaction between CIH and CTD. Since this glycine seems to be conserved among distant species like humans, D.melanogaster, and C.elegansother species, it could be important in NALCN channelosome function19,20. Although this paper provides a case with IHPRF1 manifestation that has a homozygous mutation in glycine 883, further functional studies are needed to investigate the mechanistic effect of this mutation on NALCN channelosome function. This article broadens the clinical range of IHPRF1 syndrome in addition to genotypic features. One of the cases, case 3, had recurrent urinary tract infections due to incomplete bladder emptying. Since the ionic current mediated by the NALCN channel regulates neuronal excitability, it could also play a role in muscle contraction21. Detrusor muscle contraction is responsible for bladder emptying, and NALCN impairment may poorly affect the contracture of the detrusor muscle and leads to incomplete emptying of the bladder and hence urinary tract infection22. Recurrent urinary tract infections had not been reported before in the literature and could expand the clinical spectrum of IHPRF1 syndrome, however further cases are needed to support this finding. Although dominant mutations in the NALCN gene, which are mostly gain-of-function mutations, lead to a well-characterized syndrome, recessive mutations cause a syndrome with a variable spectrum of symptoms. Therefore, identification and presentation of cases with IHPRF1 syndrome could further expand this disease's clinical spectrum. Due to the variable symptoms of IHPRF1 syndrome, WES is the primary diagnostic modality in this disease; however, by improving the definition of IHPRF1 syndrome, physicians could emphasize NALCN gene mutation screening in their differential diagnosis. In conclusion, the study adds three additional homozygote variations in the NALCN gene, expanding the clinical range of IHPRF1 syndrome by incorporating recurrent urinary tract infections as well as the genotypic spectrum of this condition. "