Assessment of pathogenicity and functional characterization of APPL1 gene mutations in diabetic patients.

BACKGROUND: Adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) plays a crucial role in regulating insulin signaling and glucose metabolism. Mutations in the APPL1 gene have been associated with the development of maturity-onset diabetes of the young type 14 (MODY14). Currently, only two mutations [c.1655T>A (p.Leu552*) and c.281G>A p.(Asp94Asn)] have been identified in association with this disease. Given the limited understanding of MODY14, it is imperative to identify additional cases and carry out comprehensive research on MODY14 and APPL1 mutations. AIM: To assess the pathogenicity of APPL1 gene mutations in diabetic patients and to characterize the functional role of the APPL1 domain. METHODS: Patients exhibiting clinical signs and a medical history suggestive of MODY were screened for the study. Whole exome sequencing was performed on the patients as well as their family members. The pathogenicity of the identified APPL1 variants was predicted on the basis of bioinformatics analysis. In addition, the pathogenicity of the novel APPL1 variant was preliminarily evaluated through in vitro functional experiments. Finally, the impact of these variants on APPL1 protein expression and the insulin pathway were assessed, and the potential mechanism underlying the interaction between the APPL1 protein and the insulin receptor was further explored. RESULTS: A total of five novel mutations were identified, including four missense mutations (Asp632Tyr, Arg633His, Arg532Gln, and Ile642Met) and one intronic mutation (1153-16A>T). Pathogenicity prediction analysis revealed that the Arg532Gln was pathogenic across all predictions. The Asp632Tyr and Arg633His variants also had pathogenicity based on MutationTaster. In addition, multiple alignment of amino acid sequences showed that the Arg532Gln, Asp632Tyr, and Arg633His variants were conserved across different species. Moreover, in in vitro functional experiments, both the c.1894G>T (at Asp632Tyr) and c.1595G>A (at Arg532Gln) mutations were found to downregulate the expression of APPL1 on both protein and mRNA levels, indicating their pathogenic nature. Therefore, based on the patient's clinical and family history, combined with the results from bioinformatics analysis and functional experiment, the c.1894G>T (at Asp632Tyr) and c.1595G>A (at Arg532Gln) mutations were classified as pathogenic mutations. Importantly, all these mutations were located within the phosphotyrosine-binding domain of APPL1, which plays a critical role in the insulin sensitization effect. CONCLUSION: This study provided new insights into the pathogenicity of APPL1 gene mutations in diabetes and revealed a potential target for the diagnosis and treatment of the disease.

Novel TNC-PDGFD fusion in fibrosarcomatous dermatofibrosarcoma protuberans: a case report.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a superficial fibroblastic tumor characterized by high rate of local recurrence and low metastatic potential. Fibrosarcomatous transformation can rarely arise in DFSP either de novo or as recurrent, which represents a form of tumor progression and carries an increased risk of metastasis over classic DFSP. Cytogenetically, DFSP is characterized by a recurrent unbalanced chromosome translocation t (17;22)(q22;q13), leading to the formation of COL1A1-PDGFB fusion transcript that is present in more than 90% of cases. Alternative fusions involving the PDGFD with partners of COL6A3 or EMILIN2 have recently been documented in less than 2% of cases. Herein, we report a DFSP with fibrosarcomtous morphology harboring a novel TNC-PDGFD fusion. CASE PRESENTATION: A 54-year-old female presented with a slowly growing mass in the right thigh. Excision demonstrated a 2-cm ovoid, well-circumscribed, gray-white, mass. Microscopic examination revealed a partially encapsulated subcutaneous nodule without dermal connection. The neoplasm was composed of cellular and fairly uniform spindle cells with brisk mitoses, arranged in elongated fascicles and herringbone patterns, with focal collagenized stroma. The neoplastic cells were positive for CD34 and smooth muscle actin. Fluorescence in-situ hybridization analyses showed negative for COL1A1-PDGFB fusion as well as NTRK1/2/3 rearrangements. A subsequent RNA sequencing detected an in-frame fusion between exon 15 of TNC and exon 6 of PDGFD. This fusion was further confirmed by nested reverse transcription polymerase chain reaction amplification followed by Sanger sequencing. A diagnosis of fibrosarcomatous DFSP was rendered and the patient was in good status at a follow-up of 12 months after the operation. CONCLUSIONS: We report a fibrosarcomatous DFSP with novel TNC-PDGFD fusion, which adds to the pathologic and genetic spectrum of PDGFD-rearranged DFSP.

TNF-α induces Drp1-mediated mitochondrial fragmentation during inflammatory cardiomyocyte injury.

Dynamin-related peptide 1 (Drpl)-mediated mitochondrial fission is an important process associated with cardiac dysfunction under different pathological conditions. The aim of the present study was to investigate the expression of Drpl during inflammatory myocardial injury. Sprague­Dawley rats were treated intraperitoneally with lipopolysaccharides (LPS). Furthermore, cultured H9C2 cardiomyocytes were treated with LPS, interleukin­6 (IL­6) and tumor necrosis factor­α (TNF­α). Total and mitochondrial proteins were isolated from the heart tissue of rats and from the H9C2 cardiomyocytes. Expression levels of Drp1 and RhoA were analyzed by western blotting. Mitochondrial morphology was determined using confocal laser microscopy. The levels of mitochondrial Drp1 and phosphorylated­Drp1 (p­Drp1) Ser616 were revealed to be increased in rats 6 h after injection with LPS (5, 10 or 20 mg/kg). Furthermore, treatment with LPS and IL­6 did not demonstrate a significant effect on the expression of total and mitochondrial Drp1 in H9C2 cardiomyocytes in vitro; however, treatment with TNF­α (20 ng/ml) significantly enhanced the levels of mitochondrial Drp1 and p­Drp1 Ser616. Following TNF­α treatment, the expression of Ras homolog gene family member A (RhoA) was also revealed to increase. Treatment with both Y­27632 and fasudil, [Rho kinase (ROCK) inhibitors], was demonstrated to attenuate the otherwise TNF­α­induced increase in p­Drp1 Ser616 and mitochondrial Drp1. In addition, it was revealed that Y­27632 and fasudil may also attenuate the TNF­α­induced increase in mitochondrial fragmentation and cell viability. Therefore, the findings of the present study suggest that TNF­α is the predominant inducer of Drp1 S616 phosphorylation during sepsis. The results of the present study also suggest that the RhoA/ROCK pathway may be involved in the phosphorylation and mitochondrial translocation of Drp1, which leads to mitochondrial fragmentation.