Aims/hypothesis

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/interpretation

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.

Data availability

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).

Graphical Abstract

中文翻译：

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RFX6 单倍体不足通过受损的 β 细胞功能诱发糖尿病

目标/假设

调节因子 X 6 (RFX6) 对于胰腺内分泌发育和分化至关重要。RFX6变体 p.His293LeufsTer7 在芬兰人群中显着富集，近 1:250 人为携带者。重要的是，FinnGen 研究表明杂合子携带者极易患 2 型糖尿病和妊娠期糖尿病。然而，这种倾向的确切机制仍不清楚。

方法

为了了解该变体在 β 细胞发育和功能中的作用，我们使用 CRISPR 技术生成等位基因系列的多能干细胞。我们创建了两种同基因干细胞模型：人类胚胎干细胞模型；以及患者来源的干细胞模型。两者均分化为胰岛谱系（干细胞衍生胰岛，SC-胰岛），然后植入免疫功能低下的 NOD-SCID-Gamma 小鼠中。

结果

纯合变体RFX6 ^−/−的干细胞模型不出所料地无法产生分泌胰岛素的胰腺 β 细胞，这反映了米切尔-莱利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，并伴有细胞凋亡增加。有趣的是，杂合的RFX6 ^+/- SC-胰岛表现出RFX6单倍体不足（蛋白质表达减少 54.2%），与 β 细胞成熟标记物减少、钙信号传导改变和胰岛素分泌受损（基础和高葡萄糖条件下减少 62% 和 54%）相关， 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。植入小鼠体内后，胰岛素分泌持续减少，这与变异携带者患糖尿病的风险增加相一致。

结论/解释

我们的等位基因系列同基因 SC 胰岛模型是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 RFX6 在增强和维持胰腺祖细胞库方面的关键作用，它具有内分泌障碍并在其丢失后增加细胞死亡。我们证明，RFX6单倍体不足不会影响 β 细胞数量或胰岛素含量，但会损害功能，使功能丧失变异的杂合携带者易患糖尿病。

数据可用性

H1 RFX6基因型胰腺分化阶段 3、5 和 7 的超深批量 RNA-seq 数据存储在 Gene Expression Omnibus 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)。

图形概要