背景:衔接蛋白,磷酸酪氨酸与PH结构域和亮氨酸拉链1(APPL1)相互作用在调节胰岛素信号传导和葡萄糖代谢中起着至关重要的作用。APPL1基因的突变与年轻14型(MODY14)的成熟发作型糖尿病的发展有关。目前,只有两个突变[c.1655T>A(p。Leu552*)和c.281G>Ap。(Asp94Asn)]已被鉴定与该疾病有关。鉴于对MODY14的了解有限,必须识别更多病例并对MODY14和APPL1突变进行全面研究。
目的:评估APPL1基因突变在糖尿病患者中的致病性,并表征APPL1结构域的功能作用。
方法:筛选显示临床体征和提示MODY病史的患者进行研究。对患者及其家庭成员进行全外显子组测序。基于生物信息学分析预测鉴定的APPL1变体的致病性。此外,通过体外功能实验初步评价了新型APPL1变异体的致病性。最后,评估了这些变体对APPL1蛋白表达和胰岛素途径的影响,并进一步探讨了APPL1蛋白与胰岛素受体相互作用的潜在机制。
结果:共鉴定出5个新突变,包括四个错义突变(Asp632Tyr,Arg633His,Arg532Gln,和Ile642Met)和一个内含子突变(1153-16A>T)。致病性预测分析显示,在所有预测中,Arg532Gln都是致病性的。Asp632Tyr和Arg633His变体也具有基于MutationTaster的致病性。此外,氨基酸序列的多重比对显示Arg532Gln,Asp632Tyr,和Arg633His变体在不同物种中保守。此外,在体外功能实验中,发现c.1894G>T(在Asp632Tyr)和c.1595G>A(在Arg532Gln)突变均在蛋白质和mRNA水平上下调APPL1的表达,表明它们的致病性。因此,根据患者的临床和家族史,结合生物信息学分析和功能实验的结果,c.1894G>T(在Asp632Tyr)和c.1595G>A(在Arg532Gln)突变被归类为致病性突变。重要的是,所有这些突变均位于APPL1的磷酸酪氨酸结合结构域内,该结构域在胰岛素增敏作用中起关键作用.
结论:这项研究为APPL1基因突变在糖尿病中的致病性提供了新的见解,并揭示了该疾病的诊断和治疗的潜在靶标。
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.
OBJECTIVE: 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.
CONCLUSIONS: 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.