背景:Peutz-Jeghers综合征(PJS)是一种罕见的遗传性肿瘤疾病,主要与丝氨酸/苏氨酸激酶11(STK11/LKB1)基因突变有关。植入前基因检测可以保护患者的后代免受突变基因的影响;然而,该基因中的一些变异已被解释为不确定意义的变异(VUS),这使得遗传咨询中的生殖决策复杂化。
目的:鉴定两种错义变异的致病性,为临床提供指导。
方法:对在中信湘雅生殖与遗传医院接受治疗的PJS患者的外周血进行全外显子组基因测序和Sanger测序。软件被用来预测蛋白质结构,养护,两个错义变异位点在PJS患者中的致病性。此外,构建质粒并转染HeLa细胞观察细胞生长。使用蛋白质印迹和免疫组织化学比较变体组和野生型组之间信号通路表达的差异。使用单向方差分析进行统计学分析。P<0.05被认为具有统计学意义。
结果:我们鉴定了两个错义STK11基因VUS[c.889A>G(p。Arg297Gly)和c.733C>T(p。Leu245Phe)]在9个寻求生殖援助的无关PJS家庭中。两个错义VUS位于丝氨酸/苏氨酸激酶的催化域,它是肝激酶B1(LKB1)蛋白的关键结构。体外实验表明,转染变异型细胞的Thr172和Ser428的LKB1磷酸化水平明显高于野生型细胞。此外,两种错义STK11变异体促进HeLa细胞增殖。随后的免疫组织化学分析显示磷酸化-AMPK(Thr172)在胃中的表达显著降低,结肠,与非PJS患者相比,PJS患者的子宫息肉具有错义变异。我们的发现表明,这两个错义STK11变体可能是致病的,并且使STK11基因失活。使其失去调节下游磷酸化AMPK(Thr172)的功能,这可能导致PJS的发展。在这两个临床特征的PJS患者中鉴定致病性突变有助于指导他们走向最合适的妊娠辅助模式。
结论:这两种错义变异可以解释为可能的致病变异,在这两名患者中介导了PJS的发作。这些发现不仅为临床决策提供了见解,但也为进一步研究和重新分析罕见疾病中的错义VUS奠定了基础。
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.
OBJECTIVE: 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.
CONCLUSIONS: 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.