Abstract：
OBJECTIVE: Regulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown.
METHODS: To understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice.
RESULTS: Stem cell models of the homozygous variant RFX6-/- predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell-Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/- SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes.
CONCLUSIONS: Our allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes.
METHODS: Ultra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 ).
METHODS: To understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice.
RESULTS: Stem cell models of the homozygous variant RFX6-/- predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell-Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/- SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes.
CONCLUSIONS: Our allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes.
METHODS: Ultra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 ).
摘要：
目的：调节因子X6（RFX6）对胰腺内分泌发育和分化至关重要。RFX6变体p.His293LeufsTer7在芬兰人口中大大丰富，以近1:250人作为载体。重要的是,FinnGen的研究表明,杂合子携带者易患2型和妊娠期糖尿病.然而,这种倾向的确切机制仍然未知。
方法：要了解该变体在β细胞发育和功能中的作用，我们使用CRISPR技术产生等位基因系列多能干细胞。我们创建了两种同基因干细胞模型：人类胚胎干细胞模型；和患者来源的干细胞模型。两者都分化为胰岛谱系（干细胞衍生的胰岛，SC-胰岛)，然后植入免疫受损的NOD-SCID-Gamma小鼠。
结果：纯合变体RFX6-/-的干细胞模型可以预见无法产生分泌胰岛素的胰腺β细胞，反映了Mitchell-Riley综合征中观察到的表型。值得注意的是,在胰腺内分泌阶段，前体标志物NEUROG3和SOX9上调,伴随细胞凋亡增加。有趣的是，杂合RFX6+/-SC-胰岛表现出RFX6单倍体不足（蛋白质表达减少54.2%），与β细胞成熟标记降低相关，钙信号改变和胰岛素分泌受损（在基础和高糖条件下减少62%和54%，分别)。然而,RFX6单倍体不足对β细胞数量或胰岛素含量没有影响。在小鼠体内植入后，胰岛素分泌的减少持续存在，与变异携带者患糖尿病的风险增加相一致。
结论：我们的等位基因系列同基因SC-胰岛模型代表了阐明人类糖尿病特定病因的强大工具，能够灵敏地检测β细胞发育和功能的畸变。我们强调了RFX6在增强和维持胰腺祖细胞池中的关键作用，内分泌障碍和细胞死亡增加。我们证明RFX6单倍体不足不影响β细胞数量或胰岛素含量，但确实损害功能，糖尿病易感功能丧失变异的杂合携带者。
方法：H1RFX6基因型的胰腺分化第3、5和7阶段的超深度批量RNA-seq数据保存在基因表达综合数据库中，登录号为GSE234289。原始蛋白质印迹图像存放在Mendeley（https://data。mendeley.com/datasets/g75drr3mgw/2)。
方法：要了解该变体在β细胞发育和功能中的作用，我们使用CRISPR技术产生等位基因系列多能干细胞。我们创建了两种同基因干细胞模型：人类胚胎干细胞模型；和患者来源的干细胞模型。两者都分化为胰岛谱系（干细胞衍生的胰岛，SC-胰岛)，然后植入免疫受损的NOD-SCID-Gamma小鼠。
结果：纯合变体RFX6-/-的干细胞模型可以预见无法产生分泌胰岛素的胰腺β细胞，反映了Mitchell-Riley综合征中观察到的表型。值得注意的是,在胰腺内分泌阶段，前体标志物NEUROG3和SOX9上调,伴随细胞凋亡增加。有趣的是，杂合RFX6+/-SC-胰岛表现出RFX6单倍体不足（蛋白质表达减少54.2%），与β细胞成熟标记降低相关，钙信号改变和胰岛素分泌受损（在基础和高糖条件下减少62%和54%，分别)。然而,RFX6单倍体不足对β细胞数量或胰岛素含量没有影响。在小鼠体内植入后，胰岛素分泌的减少持续存在，与变异携带者患糖尿病的风险增加相一致。
结论：我们的等位基因系列同基因SC-胰岛模型代表了阐明人类糖尿病特定病因的强大工具，能够灵敏地检测β细胞发育和功能的畸变。我们强调了RFX6在增强和维持胰腺祖细胞池中的关键作用，内分泌障碍和细胞死亡增加。我们证明RFX6单倍体不足不影响β细胞数量或胰岛素含量，但确实损害功能，糖尿病易感功能丧失变异的杂合携带者。
方法：H1RFX6基因型的胰腺分化第3、5和7阶段的超深度批量RNA-seq数据保存在基因表达综合数据库中，登录号为GSE234289。原始蛋白质印迹图像存放在Mendeley（https://data。mendeley.com/datasets/g75drr3mgw/2)。
