KIT cDNA Research Articles

Mutation analysis of gastrointestinal stromal tumors using RNA obtained via endoscopic ultrasound-guided fine-needle aspiration.

Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA) is useful for pathologically diagnosing gastrointestinal stromal tumor (GIST) before surgery. However, its role in mutation analysis remains unclear. To examine the feasibility of analyzing GIST mutations using mRNA obtained with EUS-FNA, we prospectively enrolled 41 patients with subepithelial lesion from which EUS-FNA was successfully acquired tissue sample. Thirty-two, 5, and 4 subepithelial lesions were diagnosed as GISTs, schwannomas, and leiomyomas, respectively. After RNA was extracted from FNA sample, RNA was converted to cDNA. Full-length sequence of the KIT cDNA amplified via the polymerase chain reaction (PCR) was successful in 31 (96.9%) out of 32 GIST and three out of 9 non-GIST (33.3%). The KIT mutation statuses of 31 GISTs in which KIT cDNA was amplified were successfully determined through directional sequencing. Furthermore, 15 of 16 surgically excised GISTs exhibited the same mutation status in both the EUS-FNA and resected samples. In vitro experiment, the minimum number of cells required to amplify full-length of KIT cDNA from RNA was one-tenth of that required to amplify KIT exon11 gene from DNA. This study clarifies that mutation analysis using RNA obtained with EUS-FNA is feasible and reliable. Moreover, our data would support that RNA-based mutation is superior to DNA-based mutation analysis in GIST.

Cloning and expression of caprine KIT gene and associations of polymorphisms with litter size

The full coding region of KIT mRNA was cloned from the caprine ovary. The results showed the caprine KIT cDNA (GenBank accession number KF364483) contained a 2925-bp open reading frame encoding a protein with 974 amino acid residues. BLAST analysis revealed that the caprine KIT protein had high similarity with that of four species: Ovis aries (99%), Bos taurus (99%), Sus scrofa (94%) and Homo sapiens (90%). The KIT mRNA expression pattern showed that KIT mRNA was expressed highly in kidney, ovary, uterus and breast. Two single nucleotide polymorphisms (g.88430T > A and g.120466G > A) in the caprine KIT gene were detected by PCR–restriction fragment length polymorphism (RFLP) and DNA sequencing in 735 goats of Xinong Saanen, Guanzhong and Boer breeds. The g.88430T > A mutation was a missense mutation (Tyr > Asn at position 409 amino acid of KIT). The association study has been done by jointly analysing all data in one analysis. The result showed that individuals with TT and TA genotypes had their litter size increased by 0.11 and 0.09, respectively, compared with those with AA genotype at the g.88430T > A locus for three goat breeds (P < 0.05). Further analysis revealed that combined genotype TTAA was better than the others for litter size in three goat breeds. Therefore, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the KIT gene could serve as a genetic marker for litter size in goat breeding.

Selecting Tyrosine Kinase Inhibitors for Gastrointestinal Stromal Tumor with Secondary KIT Activation-Loop Domain Mutations

Advanced gastrointestinal stromal tumors (GIST), a KIT oncogene-driven tumor, on imatinib mesylate (IM) treatment may develop secondary KIT mutations to confer IM-resistant phenotype. Second-line sunitinib malate (SU) therapy is largely ineffective for IM-resistant GISTs with secondary exon 17 (activation-loop domain) mutations. We established an in vitro cell-based platform consisting of a series of COS-1 cells expressing KIT cDNA constructs encoding common primary±secondary mutations observed in GISTs, to compare the activity of several commercially available tyrosine kinase inhibitors on inhibiting the phosphorylation of mutant KIT proteins at their clinically achievable plasma steady-state concentration (Css). The inhibitory efficacies on KIT exon 11/17 mutants were further validated by growth inhibition assay on GIST48 cells, and underlying molecular-structure mechanisms were investigated by molecular modeling. Our results showed that SU more effectively inhibited mutant KIT with secondary exon 13 or 14 mutations than those with secondary exon 17 mutations, as clinically indicated. On contrary, at individual Css, nilotinib and sorafenib more profoundly inhibited the phosphorylation of KIT with secondary exon 17 mutations and the growth of GIST48 cells than IM, SU, and dasatinib. Molecular modeling analysis showed fragment deletion of exon 11 and point mutation on exon 17 would lead to a shift of KIT conformational equilibrium toward active form, for which nilotinib and sorafenib bound more stably than IM and SU. In current preclinical study, nilotinib and sorafenib are more active in IM-resistant GISTs with secondary exon 17 mutation than SU that deserve further clinical investigation.

Gene Expression Patterns of Hemizygous and Heterozygous KIT Mutations Suggest Distinct Oncogenic Pathways: A Study in NIH3T3 Cell Lines and GIST Samples

ObjectiveMost gain of function mutations of tyrosine kinase receptors in human tumours are hemizygous. Gastrointestinal stromal tumours (GIST) with homozygous mutations have a worse prognosis. We aimed to identify genes differentially regulated by hemizygous and heterozygous KIT mutations.Materials and MethodsExpression of 94 genes and 384 miRNA was analysed with low density arrays in five NIH3T3 cell lines expressing the full-length human KIT cDNA wild-type (WT), hemizygous KIT mutation with del557-558 (D6) or del564-581 (D54) and heterozygous WT/D6 or WT/D54. Expression of 5 of these genes and 384 miRNA was then analysed in GISTs samples.ResultsUnsupervised and supervised hierarchical clustering of the mRNA and miRNA profiles showed that heterozygous mutants clustered with KIT WT expressing cells while hemizygous mutants were distinct. Among hemizygous cells, D6 and D54 expressing cells clustered separately. Most deregulated genes have been reported as potentially implicated in cancer and severals, as ANXA8 and FBN1, are highlighted by both, mRNA and miRNA analyses. MiRNA and mRNA analyses in GISTs samples confirmed that their expressions varied according to the mutation of the alleles. Interestingly, RGS16, a membrane protein of the regulator of G protein family, correlate with the subcellular localization of KIT mutants and might be responsible for regulation of the PI3K/AKT signalling pathway.ConclusionPatterns of mRNA and miRNA expression in cells and tumours depend on heterozygous/hemizygous status of KIT mutations, and deletion/presence of TYR568 & TYR570 residues. Thus each mutation of KIT may drive specific oncogenic pathways.

A mutation in the Kit gene leads to novel gonadal phenotypes in both heterozygous and homozygous mice

The direct sequencing of the Kit cDNA obtained from mutant mice was used to reveal the molecular nature of the W(-3Bao) ENU-induced mutation. There was a T to A transversion at the 441st nucleotide in the W(-3Bao) open reading frame (ORF), which introduced a pre-mature termination codon at residue 147. The gross embryonic development, hematopoiesis and spermatogenesis were examined in the mutant mice. There was no visible difference among the W(-3Bao/+), W(-3Bao/3Bao) and wild type embryos before embryonic day 12.5. W(-3Bao/3Bao) embryos appeared pale after E14.5 and dwarf after E16.5. An extremely low level of hematochrome and large red blood cells were found in W(-3Bao/3Bao) 18.5 days old embryos, leading to the stillbirth of the homozygotes. In 18.5 days old embryos the spermatogonia of W(-3Bao/3Bao) embryos did not migrate to the contorted seminiferous tubules properly, but instead were found in the interstitial tissue. The spermatogonia of W(-3Bao/+) or W(+/+) mice were present in both the interstitial tissue and contorted seminiferous tubules. In the adult male hetereozygotes, there are contorted seminiferous tubules with no spermatogonia, suggesting that the migration defect was dominant. In female W(-3Bao/3Bao) ovaries, primordial follicles were absent while primordial follicles appeared clearly in the ovaries of W(-3Bao/+) or W(+/+) mice. With a nonsense mutation in the Kit gene, W(-3Bao/+) mice show white spotting and an abnormal development of the contorted seminiferous tubules and W(-3Bao/3Bao) mice are stillborn due to severe macrocytic anemia, and have abnormal genital glands in both the male and female.

Homozygous lethality and heterozygous spotting due to a novel missense mutation in the mouse Kit gene

Abstract N-ethyl-N-nitrosourea (ENU) mutagenesis in mice can be used to study gene function in vivo and to establish genetic mouse models of human disease. In this study, a white spotted mouse (named KitW-1 Bao) was obtained by ENU-induced mutagenesis. Inheritance testing showed a single-gene dominant mutation and lethality in the KitW-1 Bao homozygous mice. The mutation was mapped to Chromosome 5 between markers D5 Mit356 and D5 Mit308. The region contains the Kit gene, whose mutations are known to lead to pigmentation defects in mice. Sequence analysis of the Kit cDNA from KitW-1 Bao heterozygotes revealed an A to T missense mutation resulting in an amino acid substitution of Asp (D) by Val (V) at amino acid position 849 within a highly conserved tyrosine kinase domain. The combined phenotype displayed by the KitW-1 Bao heterozygous and homozygous mutant mice demonstrates the critical function of the highly conserved aspartic acid residue at position 849 in the Kit gene product.

KIT Mutations Induce Intracellular Retention and Activation of an Immature Form of the KIT Protein in Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GIST) are frequently associated with gain-of-function mutations of KIT, which can be inhibited by imatinib both in vitro and in vivo. The survival of patients with GIST, following imatinib therapy, has been correlated with the nature of mutations but not with KIT expression. Subcellular localization, activation, and trafficking of the mature and the immature forms of KIT were investigated in GIST samples and in NIH3T3 cells infected with two different GIST-type exon 11-mutated human KIT cDNA. Paranuclear dot expression of KIT was more frequent in GISTs with homozygous KIT mutations than in those with heterozygous (P = 0.01) or no mutations (P < 0.01). Activation of the immature 125 kDa form of KIT was detected in most GISTs with KIT mutations but not in GISTs without KIT mutations. In NIH3T3 cells, mutant KIT was mainly retained within endoplasmic reticulum and Golgi compartments in an immature constitutively phosphorylated form, whereas the wild-type KIT was expressed at the plasma membrane, in a mature nonphosphorylated form. Imatinib-induced inhibition of the phosphorylation of immature and mature mutant KIT proteins resulted in the restoration of KIT expression at the cell surface. These results show that GIST-type KIT mutations induce an activation-dependent alteration of normal maturation and trafficking, resulting in the intracellular retention of the activated kinase within the cell. These observations likely account for the absence of correlation between response to imatinib and KIT expression using immunohistochemistry and may deserve to be investigated in other tyrosine kinase-activated tumors.

Gain-of-function mutations of platelet-derived growth factor receptor α gene in gastrointestinal stromal tumors

Background & Aims : Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of c- kit receptor tyrosine kinase (KIT) gene, but some GISTs do not. We investigated the cause of GISTs without KIT mutations. Because GISTs apparently expressed platelet-derived growth factor receptor (PDGFR) α, we examined whether GISTs without KIT mutations had a mutation of PDGFR α. Methods : Whole coding region of PDGFR α complementary DNA (cDNA) was sequenced in GISTs with or without KIT mutations. Mutant PDGFR α cDNA was transfected into 293T human embryonic kidney cells, and autophosphorylation of PDGFR α was examined. Proliferation of Ba/F3 murine lymphoid cells stably transfected with mutant PDGFR α cDNA was estimated by tritium thymidine incorporation. Wild-type KIT cDNA was cotransfected with mutant PDGFR α cDNA, and immunoprecipitation by anti-KIT antibody was performed. Inhibitory effect of Imatinib mesylate on activated PDGFR α was examined. Results : We found 2 types of constitutively activated mutations of PDGFR α, Val-561 to Asp or Asp-842 to Val, in 5 of 8 GISTs without KIT mutations but not in 10 GISTs with KIT mutations. Stable transfection of each mutation induced autonomous proliferation of Ba/F3 cells. Constitutively activated mutant PDGFR α bound and activated the cotransfected wild-type KIT. The constitutive activation of PDGFR α with Val-561 to Asp was inhibited effectively by Imatinib mesylate but that of PDGFR α with Asp-842 to Val was inhibited only weakly, even at the concentration of 10 μmol/L. Conclusions : The gain-of-function mutations of PDGFR α appear to play an important role in development of GISTs without KIT mutations.

Male hybrid sterility of mice with the genomic region of the KitW mutation and the KitS allele from Mus spretus.

Two congenic strains, C57BL-KitW and C57BL-KitS, were generated. The KitW allele originated from strain WB-KitW and the KitS allele from Mus spretus. The KitW/KitS males showed hybrid sterility with small testes, but the females were fertile. The development of the seminiferous tubules of KitW/KitS males stopped before the spermatocyte stage and they were almost free of sperm. The Kit gene is located at position 42 on chromosome 5. We investigated in the C57BL-KitS congenic strain which part of the chromosomal region adjacent to the KitS allele is introduced from SPR into a C57BL background. The region between positions 42 and 44 was derived from SPR. Eleven amino acid substitutions of the KitS cDNA were detected by comparison with the sequence data of the +Kit cDNA from C57BL; seven were in the extracellular domain, one in the transmembrane domain, two in the kinase I domain, and one in the carboxy-terminal tail. The Kit mRNA derived from both KitW and KitS alleles was expressed in the sterile testes of KitW/KitS males.

KIT Extracellular and Kinase Domain Mutations in Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms arising in the gastrointestinal tract. GISTs express the KIT receptor tyrosine kinase, and many cases have activating mutations in the KIT juxtamembrane region. We now report an analysis of KIT cDNA and genomic sequences in eight GISTs that lack juxtamembrane region mutations. Six cases contained heterozygous exon 9 mutations in which six nucleotides, encoding Ala-Tyr, were duplicated. The other two cases contained homozygous exon 13 missense mutations, resulting in substitution of Glu for Lys(642), that were associated with constitutive KIT tyrosine phosphorylation. Sequence analysis of DNAs from nonneoplastic companion tissues revealed that both the exon 9 and exon 13 mutations were somatic. These are the first descriptions, in any tumor, of mutations in KIT exons encoding the C-terminal end of the extracellular domain and the first part of the split kinase domain. These findings indicate that KIT may be activated by mutations in at least three domains-extracellular, juxtamembrane, and kinase-in GISTs.

Close association between sequence polymorphism in the KIT gene and the roan coat color in horses.

The roan coat color in horses is controlled by a dominant allele that is lethal in the homozygous condition. Phenotypic similarities to some pigmentation disorders in human and mouse, combined with comparative mapping data, identified KIT, encoding the mast cell growth factor receptor, as a major candidate gene for the roan locus (Rn). Rn has previously been mapped to equine linkage group (LG) II. In this study, LGII was expanded with KIT and PDGFRA (platelet-derived growth factor receptor alpha) by use of RFLP and linkage analysis. Moreover, highly significant linkage disequilibrium between Rn and a KIT TaqI RFLP, representing a synonymous substitution in exon 19, was revealed. There was a strong KIT-Rn association in most breeds. Almost the complete KIT-encoding sequence was determined by sequence analysis of RT-PCR products. Comparison of horse KIT cDNA sequences, representing three different alleles (two different rn and one Rn), revealed five sequence polymorphisms and several mRNA splice variants, but none of these proved to be specifically associated with Rn. An insertion of a partial (79 bp) LINE1-element between exons 1 and 2, leading to a frameshift, represented about 30% of KIT transcripts in the Belgian roan horse used for the sequence analysis. However, an association between this L1 splice insertion and the roan phenotype was not verified when testing additional unrelated roan and non-roan horses from different breeds. The study strengthens the hypothesis that the roan coat color is controlled by KIT, but further analyses are needed to reveal the causative mutation(s).

A novel gain-of-function mutation of c- kit gene in gastrointestinal stromal tumors

Background & Aims: The c- kit gene encodes a receptor tyrosine kinase (KIT). Recently, we found gain-of-function mutations of the c- kit gene in gastrointestinal stromal tumors (GISTs). All mutations were confined within the 11 amino acids (Lys-550 to Val-560) in the juxtamembrane domain, but one GIST showed a novel deletion-type mutation at codon 579 (Asp) in the juxtamembrane domain. The aim of this study was to clarify whether the mutation is activating. Methods: Mutant c- kit cDNA was transfected into an interleukin 3 (IL-3)-dependent Ba/F3 murine lymphoid cell line, and the magnitude of autophosphorylation of the mutant KIT was examined with or without stem cell factor (SCF), a ligand of KIT. An in vitro kinase assay was also performed. The biological behavior of the transfectant was estimated by both an in vitro proliferation assay and in vivo transplantation to nude mice. Results: The mutant KIT exhibited constitutive phosphorylation and strong kinase activity without SCF. The transfectant grew autonomously without IL-3 and SCF, and it formed tumors in nude mice. Conclusions: Deletion at codon 579 (Asp) in the juxtamembrane domain of the c- kit gene is a novel gain-of-function mutation other than the region between Lys-550 and Val-560. GASTROENTEROLOGY 1998;115:1090-1095

Recessive spotting: a linked locus that interacts with W/Kit but is not allelic.

The murine coat-colour mutation recessive spotting (rs) maps very closely to the W/Kit locus, encoding the proto-oncoprotein Kit, the protein tyrosine kinase receptor for stem cell factor. Kit is important in the development of melanocytes, germ cells, interstitial cells of Cajal (ICC) and haemopoietic lineages, including mast cells. rs has never been genetically separated from Kit, and interacts with Kit mutations, suggesting that it is a recessive allele of Kit. Here we have tested this possibility. We have shown previously that diploid rs/rs melanocytes proliferated more slowly than did +/+ melanocytes, as did an immortal line of rs/rs melanocytes, melan-rs. The Kit mRNA level in rs/rs melanocytes was indistinguishable from that of other melanocyte lines. The Kit cDNA sequence from rs/rs melanocytes and the kinase activity of Kit in rs/rs mast cells appeared to be normal. No deficiency of mast cells or ICC was observed in rs/rs mice. Moreover, following the overexpression of a normal Kit cDNA, proliferation of rs/rs melanocytes was retarded further, but that of +/+ melanocytes was increased, indicating an intracellular interaction between rs and Kit. Of other closely linked tyrosine kinase genes, melanocytes and melanoblasts did not express mRNA for Pdgfra, Flk-1 or Txk, but both expressed Tec, encoding a nonreceptor kinase that interacts with Kit. rs is not a mutation in Kit, although we have confirmed that rs interacts with Kit. It seems unlikely that rs affects Pdgfra, Flk-1 or Txk, but Tec remains a candidate for rs.

Cloning and subcellular localization of hamster and rat isopentenyl diphosphate dimethylallyl diphosphate isomerase. A PTS1 motif targets the enzyme to peroxisomes.

To date, isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase; EC 5.3.3.2) is presumed to have a cytosolic localization. However, we have recently shown that in permeabilized cells lacking cytosolic components, mevalonate can be converted to cholesterol, implying that all of the enzymes required for the conversion of mevalonate to farnesyl diphosphate are found in the peroxisome. To provide unequivocal evidence for the subcellular localization of IPP isomerase, in this study, we have cloned the rat and hamster homologues of IPP isomerase and identified the signal that targets this enzyme to peroxisomes. In addition, we also demonstrate that IPP isomerase is regulated at the mRNA level.