Due to its essential role of suppressing KIT proto-oncoprotein, the juxtamembrane (JM) domain releases its brake, when mutated, leading to proliferation promotion and apoptosis inhibition [3, 7]. This involvement of the JM domain-encoding exon 11 in GIST development is highly revealed by an average 86% mutation rate among KIT-mutated GIST patients [22,23,24,25]. In this study, exon 11 was mutated in about 58% of GIST patients approximating the average worldwide rate (60%) that spreads over the range 22–90% in more than a hundred studies. In discord with inherited variations, these reported frequencies of exon 11 somatic mutations among GIST patients were not correlated with geographic region according to our statistical estimation (data not shown). Nucleotide deletions and substitutions were the only mutation types detected in our GIST patients (Table 1); this accords with the majority of publications where deletions and substitutions are considerably reported contrary to insertions and duplications [26, 27]. In discord with numerous studies [2, 28], however, the combination of deletion-substitution mutation type was highly detected in our study group (Table 1).

Our bioinformatic analysis by SIFT, PolyPhen-2, fathmm, and MutationTaster tools revealed that all seven deletions and nine amino acid substitutions encountered in the JM domain were pathogenic (Tables 2 and 3). Despite their sole discordant predictions between the four tools, we suppose that Val555Ile and Arg586Lys mutations are more likely pathogenic according to the pathogenicity weight, conservation index, and large diverse databases adopted by relevant tools. Hence, the occurrence of any of these sixteen mutations in the KIT proto-oncogene might promote tumorigenesis. Their pathogenicity is further emphasized as high tumor grade seemed independent of type and number of mutations encountered in the JM domain of the KIT gene in our GIST patients (p > 0.05).

This virtual outcome is underlined by X-ray crystallographic reports that demonstrated the key role of the JM domain in general and its critical codons Tyr553, Trp557, Val559, Val560, and from Tyr547 to Gly565 in particular [29]. Notably, these aforementioned codons were affected by most presumably pathogenic mutations detected in our GIST patients, possibly disrupting JM function in stabilizing autoinhibited KIT and leading to cancer development [30, 31]. Moreover, the severity of these gain-of-function mutations in the JM domain, whether being substitutions or deletions, has already been manifested by in vitro and in vivo experimental evidence [30, 31]. Besides folding autonomously, the JM domain has been found to be highly conserved in class III receptor tyrosine kinases (RTKs) family. In fact, mutant JM domain demonstrated disordered secondary structure, reduced binding affinity to the kinase domain, less effective inhibition of receptor catalytic activity, and extremely rapid kinetics of ligand-independent receptor activation. Ultimately, the existence of substitution or deletion mutations in the JM domain of the KIT protein seemed sufficient for cell transformation [31].

Obviously, deletion mutations were specifically severe and seem to lower survival rate; this is supported by the following:

(i) Their high frequency in our cases with mutant JM domain (21 of 28; 75%).

(ii) Their apparent negative selection in favor of substitution mutations in our elderly GIST patients.

(iii) In addition to clinical evidence inferred by more than a dozen studies [8, 32, 33]

Interestingly, Trp557_Lys558del mutation was the most frequently encountered deletion (14 of 21; 67%). Furthermore, deletion mutations affected both codons 557 and 558 in 90% (19 of 21) of our cases. These observations point to the phenomenal severity of deletions in these amino acid positions.

GIST patients with mutated JM domain have significantly higher response rates to imatinib therapy [1, 9, 34, 35]; this was evident in seven of our patients including four responders with exon 11 mutations and three nonresponders with wild-type exon 11. Nevertheless, therapy information for the remaining GIST patients was not retrievable; hence, the association between imatinib response and the JM domain mutations could not be analyzed.

Our data indicated an association between high tumor grade and age above 60 (p < 0.05), an observation likely attributed to the accumulation of additional mutations over a longer lifespan in other loci of the genome. This explains the absence of association between tumor grade and the occurrence of JM domain mutations in our GIST patients (p > 0.05). Obviously, gender might not contribute to the tumor pathogenicity as it appeared independent of the occurrence, type, and number of JM domain mutations as well as tumor grade and age of onset. Nevertheless, it has been previously observed that men are slightly more likely to develop the disease and to have a worse prognosis than women [36, 37].

Extending the catalogue of KIT mutations in GIST, eight novel additional variations have been detected in our patients including two in intron 10, three in exon 11, and three in intron 11. Intriguingly, the two novel substitutions in Tyr553 and Lys581 occurred in one case and were deemed pathogenic by all utilized bioinformatic tools. This consists with the significance of Tyr553 amino acid in inhibiting KIT by interacting with Asp810 and Glu640 of kinase domain [29]. The third novel virtually pathogenic single-nucleotide deletion in exon 11 shifted the reading frame and incorporated a premature stop codon perhaps leading to the production of a truncated protein. The novel intron variations were supposedly neutral via in silico analysis except the one encountered twice and located within a splicing motif. These findings might not be conclusive as enrolling healthy individuals was beyond the scope of our study. Further biological and clinical studies might reveal the impact of these novel variations. Otherwise, genome-wide association studies in the future might classify such presumably neutral variants as polymorphisms.