BACKGROUND Peutz-Jeghers syndrome(PJS)is a rare hereditary neoplastic disorder mainly associated with serine/threonine kinase 11(STK11/LKB1)gene mutations.Preimplantation genetic testing can protect a patient’s offsp...BACKGROUND Peutz-Jeghers syndrome(PJS)is a rare hereditary neoplastic disorder mainly associated with serine/threonine kinase 11(STK11/LKB1)gene mutations.Preimplantation genetic testing can protect a patient’s offspring from mutated genes;however,some variations in this gene have been interpreted as variants of uncertain significance(VUS),which complicate reproductive decision-making in genetic counseling.AIM To identify the pathogenicity of two missense variants and provide clinical guidance.METHODS Whole exome gene sequencing and Sanger sequencing were performed on the peripheral blood of patients with PJS treated at the Reproductive and Genetic Hospital of Citic-Xiangya.Software was employed to predict the protein structure,conservation,and pathogenicity of the two missense variation sites in patients with PJS.Additionally,plasmids were constructed and transfected into HeLa cells to observe cell growth.The differences in signal pathway expression between the variant group and the wild-type group were compared using western blot and immunohistochemistry.Statistical analysis was performed using one-way analysis of variance.P<0.05 was considered statistically significant.RESULTS We identified two missense STK11 gene VUS[c.889A>G(p.Arg297Gly)and c.733C>T(p.Leu245Phe)]in 9 unrelated PJS families who were seeking reproductive assistance.The two missense VUS were located in the catalytic domain of serine/threonine kinase,which is a key structure of the liver kinase B1(LKB1)protein.In vitro experiments showed that the phosphorylation levels of adenosine monophosphate-activated protein kinase(AMPK)at Thr172 and LKB1 at Ser428 were significantly higher in transfected variation-type cells than in wild-type cells.In addition,the two missense STK11 variants promoted the proliferation of HeLa cells.Subsequent immunohistochemical analysis showed that phosphorylated-AMPK(Thr172)expression was significantly lower in gastric,colonic,and uterine polyps from PJS patients with missense variations than in non-PJS patients.Our findings indicate that these two missense STK11 variants are likely pathogenic and inactivate the STK11 gene,causing it to lose its function of regulating downstream phosphorylated-AMPK(Thr172),which may lead to the development of PJS.The identification of the pathogenic mutations in these two clinically characterized PJS patients has been helpful in guiding them toward the most appropriate mode of pregnancy assistance.CONCLUSION These two missense variants can be interpreted as likely pathogenic variants that mediated the onset of PJS in the two patients.These findings not only offer insights for clinical decision-making,but also serve as a foundation for further research and reanalysis of missense VUS in rare diseases.展开更多
Background:Metformin has pleiotropic effects beyond glucose reduction,including tumor inhibition and immune regulation.It enhanced the anti-tumor effects of programmed cell death protein 1(PD-1)inhibitors in serine/th...Background:Metformin has pleiotropic effects beyond glucose reduction,including tumor inhibition and immune regulation.It enhanced the anti-tumor effects of programmed cell death protein 1(PD-1)inhibitors in serine/threonine kinase 11(STK11)mutant non-small cell lung cancer(NSCLC)through an axis inhibition protein 1(AXIN1)-dependent manner.However,the alterations of tumor metabolism and metabolites upon metformin administration remain unclear.Methods:We performed untargeted metabolomics using liquid chromatography(LC)-mass spectrometry(MS)/MS system and conducted cell experiments to verify the results of bioinformatics analysis.Results:According to the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database,most metabolites were annotated into metabolism,including nucleotide metabolism.Next,the differentially expressed metabolites in H460(refers to H460 cells),H460_met(refers to metformin-treated H460 cells),and H460_KO_met(refers to metformin-treated Axin1-/-H460 cells)were distributed into six clusters based on expression patterns.The clusters with a reversed expression pattern upon metformin treatment were selected for further analysis.We screened out metabolic pathways through KEGG pathway enrichment analysis and found that multiple nucleotide metabolites enriched in this pathway were upregulated.Furthermore,these metabolites enhanced the cytotoxicity of activated T cells on H460 cells in vitro and can activate the stimulator of the interferon genes(STING)pathway independently of AXIN1.Conclusion:Relying on AXIN1,metformin upregulated multiple nucleotide metabolites which promoted STING signaling and the killing of activated T cells in STK11 mutant NSCLC,indicating a potential immunotherapeutic strategy for STK11 mutant NSCLC.展开更多
目的探讨黑斑息肉综合征(PJS)的临床及其致病基因STK11特点。方法回顾分析4例PJS患儿的临床资料及患儿和父母的STK11基因突变情况。结果 4例中女3例、男1例,年龄1岁8个月至9岁6个月。均在口唇和/或手指处发现黑斑,肠镜检查均发现不同位...目的探讨黑斑息肉综合征(PJS)的临床及其致病基因STK11特点。方法回顾分析4例PJS患儿的临床资料及患儿和父母的STK11基因突变情况。结果 4例中女3例、男1例,年龄1岁8个月至9岁6个月。均在口唇和/或手指处发现黑斑,肠镜检查均发现不同位置的胃肠道多发息肉;行息肉切除术后,病理结果显示黏膜肌层的肌纤维增生形成树枝样结构。基因检测发现,4例患儿均存在STK11基因杂合突变,突变位点分别为c.582C>A,c.580G>A,c.719C>G以及c.879 ins A突变。结论基因检测有助于早期诊断PJS,STK11基因c.879 ins A突变为国内外未报道的新突变。展开更多
基金Supported by the Natural Science Foundation of Hunan Province,China,No.2023JJ30422.
文摘BACKGROUND Peutz-Jeghers syndrome(PJS)is a rare hereditary neoplastic disorder mainly associated with serine/threonine kinase 11(STK11/LKB1)gene mutations.Preimplantation genetic testing can protect a patient’s offspring from mutated genes;however,some variations in this gene have been interpreted as variants of uncertain significance(VUS),which complicate reproductive decision-making in genetic counseling.AIM To identify the pathogenicity of two missense variants and provide clinical guidance.METHODS Whole exome gene sequencing and Sanger sequencing were performed on the peripheral blood of patients with PJS treated at the Reproductive and Genetic Hospital of Citic-Xiangya.Software was employed to predict the protein structure,conservation,and pathogenicity of the two missense variation sites in patients with PJS.Additionally,plasmids were constructed and transfected into HeLa cells to observe cell growth.The differences in signal pathway expression between the variant group and the wild-type group were compared using western blot and immunohistochemistry.Statistical analysis was performed using one-way analysis of variance.P<0.05 was considered statistically significant.RESULTS We identified two missense STK11 gene VUS[c.889A>G(p.Arg297Gly)and c.733C>T(p.Leu245Phe)]in 9 unrelated PJS families who were seeking reproductive assistance.The two missense VUS were located in the catalytic domain of serine/threonine kinase,which is a key structure of the liver kinase B1(LKB1)protein.In vitro experiments showed that the phosphorylation levels of adenosine monophosphate-activated protein kinase(AMPK)at Thr172 and LKB1 at Ser428 were significantly higher in transfected variation-type cells than in wild-type cells.In addition,the two missense STK11 variants promoted the proliferation of HeLa cells.Subsequent immunohistochemical analysis showed that phosphorylated-AMPK(Thr172)expression was significantly lower in gastric,colonic,and uterine polyps from PJS patients with missense variations than in non-PJS patients.Our findings indicate that these two missense STK11 variants are likely pathogenic and inactivate the STK11 gene,causing it to lose its function of regulating downstream phosphorylated-AMPK(Thr172),which may lead to the development of PJS.The identification of the pathogenic mutations in these two clinically characterized PJS patients has been helpful in guiding them toward the most appropriate mode of pregnancy assistance.CONCLUSION These two missense variants can be interpreted as likely pathogenic variants that mediated the onset of PJS in the two patients.These findings not only offer insights for clinical decision-making,but also serve as a foundation for further research and reanalysis of missense VUS in rare diseases.
基金People’s Hospital of Xuyong County-Southwest Medical University Science and Technology Strategic Cooperation Project(2023XYXNYD05)Guangdong Association of Clinical Trials(GACT)/Chinese Thoracic Oncology Group(CTONG)and Guangdong Provincial Key Lab of Translational Medicine in Lung Cancer(2017B030314120)Natural Science Foundation of Chongqing Municipality(CSTB2023NSCQ-MSX0554).
文摘Background:Metformin has pleiotropic effects beyond glucose reduction,including tumor inhibition and immune regulation.It enhanced the anti-tumor effects of programmed cell death protein 1(PD-1)inhibitors in serine/threonine kinase 11(STK11)mutant non-small cell lung cancer(NSCLC)through an axis inhibition protein 1(AXIN1)-dependent manner.However,the alterations of tumor metabolism and metabolites upon metformin administration remain unclear.Methods:We performed untargeted metabolomics using liquid chromatography(LC)-mass spectrometry(MS)/MS system and conducted cell experiments to verify the results of bioinformatics analysis.Results:According to the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database,most metabolites were annotated into metabolism,including nucleotide metabolism.Next,the differentially expressed metabolites in H460(refers to H460 cells),H460_met(refers to metformin-treated H460 cells),and H460_KO_met(refers to metformin-treated Axin1-/-H460 cells)were distributed into six clusters based on expression patterns.The clusters with a reversed expression pattern upon metformin treatment were selected for further analysis.We screened out metabolic pathways through KEGG pathway enrichment analysis and found that multiple nucleotide metabolites enriched in this pathway were upregulated.Furthermore,these metabolites enhanced the cytotoxicity of activated T cells on H460 cells in vitro and can activate the stimulator of the interferon genes(STING)pathway independently of AXIN1.Conclusion:Relying on AXIN1,metformin upregulated multiple nucleotide metabolites which promoted STING signaling and the killing of activated T cells in STK11 mutant NSCLC,indicating a potential immunotherapeutic strategy for STK11 mutant NSCLC.
文摘目的探讨黑斑息肉综合征(PJS)的临床及其致病基因STK11特点。方法回顾分析4例PJS患儿的临床资料及患儿和父母的STK11基因突变情况。结果 4例中女3例、男1例,年龄1岁8个月至9岁6个月。均在口唇和/或手指处发现黑斑,肠镜检查均发现不同位置的胃肠道多发息肉;行息肉切除术后,病理结果显示黏膜肌层的肌纤维增生形成树枝样结构。基因检测发现,4例患儿均存在STK11基因杂合突变,突变位点分别为c.582C>A,c.580G>A,c.719C>G以及c.879 ins A突变。结论基因检测有助于早期诊断PJS,STK11基因c.879 ins A突变为国内外未报道的新突变。
