To collect all reported PTS gene variants reported in Iran and analyze their pathogenicity, a literature search was performed and a total of 54 variants were identified (Table 1). So far, only a small number of studies on the genetics of patients with PTPS deficiency have been performed in Iran, and only 800 healthy individuals have been studied in the Iranome project. On the other hand, Iran is a country with a population of about 85 million people as well as a high ethnic diversity [11,12,13,14,15,16,17,18]. In fact, recent studies have shown high mutational diversity of single-gene disorders in Iran [19,20,21,22,23]. From all the above issues, it is possible that the number of PTS gene variants in Iran increase in the future.
A total of 19 variants had been reported among Iranian patients with PTPS deficiency. c.155A>G (p.Asn52Ser) and c.317C>T (p.Thr106Met) were the most frequent variants, each with a frequency of 10% (Table 1). c.155A>G (p.Asn52Ser) has been known as a common variant in East Asia [1], especially in Taiwan [24]. In its homozygous form, c.155A>G (p.Asn52Ser) causes sever PTPS deficiency [25]. On the other hand, c.317C>T (p.Thr106Met) has a high frequency of 32% in Russia [26]. Both of these variants were reported repeatedly in combination with P/LP variants, as well as in the form of homozygous [5, 6, 24,25,26,27,28,29,30,31]. Therefore, the two ACMG-AMP criteria, PM3_VS and PP4, were assigned for these variants. Although no functional studies were found in the literature for these variants, in silico analysis revealed that both are deleterious in all 10 predictive tools, therefore, PP3 was met (Table 2). Finally, based on the assigned criteria, c.155A>G (p.Asn52Ser) and c.317C>T (p.Thr106Met) were classified as P (Table 4).
c.84-3C>G, with a frequency of 7.5%, was the third most frequent variant in Iranian patients with PTPS deficiency (Table 1). It seems that this is the highest frequency reported to date for this variant. c.84-3C>G was reported for the first time in 1997 [32]. Subsequently, it was detected with low frequencies in homozygous form or in combination with P/LP variants in at least five other studies (consistent with PM3_VS and PP4 criteria) [5, 6, 26, 31, 33]. In silico analysis of this variant showed loss of function or a strong decrease of score for authentic ASS; accordingly, exon skipping was predicted to be the final outcome (Fig. 2 and Table 3). However, according to Oppliger et al. functional analysis on this variant [32], the use of a cryptic splicing site would result in a 12 nucleotides deletion at the beginning of exon 2 and finally, a deletion of four amino acids (p.Lys29_Ser32del) (Fig. 2). These authors also reported a complete inactivity of PTPS enzyme encoded by this allele (consistent with PS3_Su criterion). Due to the assigning of PS3, PP3 was not applied for c.84-3C>G [9]. As shown in Table 4, this variant was classified as P according to ACMG-AMP guidelines.
c.281A>T (p.Asp94Val), c.331G>A (p.Ala111Thr, rs1367077861), and c.351C>A (p.Asn117Lys) had the allele frequencies of 5, 3.3, and 3.3% among Iranian PTPS patients. These variants were only reported in homozygous form and to the best of our knowledge, there is no report of their combination with P/LP variants in the literature [5, 6, 24, 34]; accordingly, along with PP4, PM3_Su was also applied [8]. Moreover, none of them were reported in gnomAD and 1KGP databases (consistent with PM2 criterion). While PP3 criterion was assigned to c.281A>T (p.Asp94Val) and c.331G>A (p.Ala111Thr) variants, c.351C>A (p.Asp117Lys) failed to reach the threshold defined by us in the Methods section (neither PP3 nor BP4 was used) (Table 2). Finally, according to ACMG-AMP guidelines, while the first two variants were classified as LP, the last one was classified as VUS (Table 4).
Nine other variants, each with a frequency of 1.67%, including c.400G>A (p.Glu134Lys, rs779681799), c.259C>T (p.Pro87Ser, rs104894276), c.286G>A (p.Asp96Asn, rs104894280), c.367C>T (p.Pro123Ser, rs141163668), c.412A>C (p.Asn138His, rs1329239489), c.293C>T (p.Pro98Leu, rs748040027), c.70C>G (p.His24Asp), c.407A>T (p.Asp136Val), and c.164-36A>G were also reported among Iranian PTPS patients. c.400G>A (p.Glu134Lys) was first identified in 2017 in an Iranian patient [5], and has subsequently been reported in Omani [30] and Russian [26] patients. Although c.259C>T (p.Pro87Ser) is a common variant in East Asia [27, 29, 35,36,37,38], it also has been detected in Iran [5] and Russia [26]. Similarly, c.286G>A (p.Asp96Asn) is common in East Asian populations [29, 35,36,37]. According to Imamura et al. study [35], the PTPS enzyme activities of p.Pro87Ser and p.Asp96Asn proteins in COS cells was 52 and 10%, respectively; therefore, PS3_Su was only applied for c.286G>A (p.Asp96Asn) (Table 4).
Oppliger et al. reported that the overexpression of c.407A>T (p.Asp136Val) in COS-1 cells would reduce PTPS activity to 31% compared to the wild-type enzyme (consistent with PS3 criterion) [32]. This variant has been found in Italian, Polish, Turkish, German, and Iranian patients so far [6, 32, 33, 39, 40]. Khatami et al. reported c.164-36A>G in homozygous form in an Iranian patient with PTPS deficiency [5] and to the best of our knowledge, there is no other report for this variant in the literature. Our analysis showed that this variant has no effect on splicing process (Table 3) and was classified as a VUS variant (Table 4). Four variants of c.200C>T (p.Thr67Met, rs370340361), c.297C>A (p.Tyr99Ter), c.163+2T>C, and c.373G>A (p.Gly125Arg) had the lowest frequencies in this study (Table 1). With the exception of c.200C>T (p.Thr67Met) [6, 32, 36, 41, 42], the other three variants have been reported in only a few studies [5, 6].
Out of 36 variants identified in the Iranome project, only three variants including c.164-1G>C, c.315-1G>A, and c.373G>A (p.Gly125Arg) were classified in the category of P/LP. c.373G>A (p.Gly125Arg) had also been reported among Iranian PTPS patients (the above paragraph) (Table 1). Both of c.164-1G>C and c.315-1G>A are rare splicing variants [26, 31]. Both of these variants cause loss of function of ASS (Table 3), however, because of assigning of PVS1 criterion, PP3 was not applied [9]. The other intronic or synonymous variants had no effect on splicing process (Tables 1 and 3) and were classified as B, LB, or VUS (Table 4). The splicing effect analysis of some of these variants are shown in Table 3.
In conclusion, the collection of all reported PTS gene variants in the Iranian population, their analysis using in silico predictive tools, and their classification according to ACMG-AMP criteria were the three main areas in the present study. The ACMG-AMP criteria need to be updated depending on the type of disease. In addition, to the best of our knowledge, no template has been described for classifying the variants identified in PTPS deficiency. Therefore, this study can be a good reference for future studies in this area.