Abstract：
The aim of this study was to perform familial co-segregation analysis and functional trial in vivo during mixed meal tolerance test (MMTT) of novel variants in diabetes candidate genes.
It is a continuation of the project \"Genetic diabetes in Lithuania\" with the cohort of 1209 patients with diabetes. Prior screening for autoimmune markers confirmed type 1 diabetes (T1D) diagnosis in 88.1% (n=1065) of patients, and targeted next-generation sequencing identified 3.5% (n=42) pathogenic variants in MODY genes. Subsequently, 102 patients were classified as having diabetes of unknown etiology. 12/102 were found to have novel variants in potential diabetes genes (RFX2, RREB1, SLC5A1 (3 patients with variants in this gene), GCKR, MC4R, CASP10, TMPRSS6, HGFAC, DACH1, ZBED3). Co-segregation analysis and MMTT were carried out in order to study beta-cell function in subjects with specific variants.
MMTT analysis showed that probands with variants in MC4R, CASP10, TMPRSS6, HGFAC, and SLC5A1 (c.1415T>C) had sufficient residual beta-cell function with stimulated C-peptide (CP) >200 pmol/L. Seven individuals with variants in RFX2, RREB1, GCKR, DACH1, ZBED3 and SLC5A1 (c.1415T>C, and c.932A>T) presented with complete beta-cell failure. No statistical differences were found between patients with sufficient CP production and those with complete beta-cell failure when comparing age at the onset and duration of diabetes. Nineteen family members were included in co-segregation analysis; no diabetes cases were reported among them. Only in patient with the variant c.1894G>A in RFX2 gene, none of the family members were affected by proband\'s variant.
Functional beta-cell study in vivo allowed to select five most probable genes for monogenic diabetes. Familial co-segregation analysis showed that novel variant in RFX2 gene could be a possible cause of diabetes. Future functional analysis in vitro is necessary to support or rule out the genetic background as a cause of diabetes.
It is a continuation of the project \"Genetic diabetes in Lithuania\" with the cohort of 1209 patients with diabetes. Prior screening for autoimmune markers confirmed type 1 diabetes (T1D) diagnosis in 88.1% (n=1065) of patients, and targeted next-generation sequencing identified 3.5% (n=42) pathogenic variants in MODY genes. Subsequently, 102 patients were classified as having diabetes of unknown etiology. 12/102 were found to have novel variants in potential diabetes genes (RFX2, RREB1, SLC5A1 (3 patients with variants in this gene), GCKR, MC4R, CASP10, TMPRSS6, HGFAC, DACH1, ZBED3). Co-segregation analysis and MMTT were carried out in order to study beta-cell function in subjects with specific variants.
MMTT analysis showed that probands with variants in MC4R, CASP10, TMPRSS6, HGFAC, and SLC5A1 (c.1415T>C) had sufficient residual beta-cell function with stimulated C-peptide (CP) >200 pmol/L. Seven individuals with variants in RFX2, RREB1, GCKR, DACH1, ZBED3 and SLC5A1 (c.1415T>C, and c.932A>T) presented with complete beta-cell failure. No statistical differences were found between patients with sufficient CP production and those with complete beta-cell failure when comparing age at the onset and duration of diabetes. Nineteen family members were included in co-segregation analysis; no diabetes cases were reported among them. Only in patient with the variant c.1894G>A in RFX2 gene, none of the family members were affected by proband\'s variant.
Functional beta-cell study in vivo allowed to select five most probable genes for monogenic diabetes. Familial co-segregation analysis showed that novel variant in RFX2 gene could be a possible cause of diabetes. Future functional analysis in vitro is necessary to support or rule out the genetic background as a cause of diabetes.
摘要：
背景：本研究的目的是在糖尿病候选基因的新型变异体的混合膳食耐受试验（MMTT）期间进行家族共分离分析和体内功能试验。
方法：这是“立陶宛遗传性糖尿病”项目的延续，该项目包括1209名糖尿病患者。在88.1%（n=1065）的患者中，自身免疫标志物的先前筛查证实了1型糖尿病（T1D）的诊断，靶向下一代测序在MODY基因中鉴定出3.5%(n=42)的致病变异。随后，102例患者被归类为病因不明的糖尿病。12/102被发现在潜在的糖尿病基因中具有新的变异（RFX2，RREB1，SLC5A1（3名患者在该基因中具有变异），GCKR,MC4R,CASP10,TMPRSS6,HGFAC,DACH1,ZBED3)。进行共分离分析和MMTT以研究具有特定变体的受试者中的β细胞功能。
结果：MMTT分析表明，在MC4R中具有变体的先证者，CASP10,TMPRSS6,HGFAC,SLC5A1（c.1415T>C）具有足够的残留β细胞功能，刺激的C肽（CP）>200pmol/L。7个个体在RFX2，RREB1，GCKR，DACH1，ZBED3和SLC5A1（c.1415T>C，和c.932A>T）表现为完全β细胞衰竭。当比较糖尿病发作时的年龄和持续时间时，在具有足够CP产生的患者和完全β细胞衰竭的患者之间没有发现统计学差异。共隔离分析包括19名家庭成员；其中没有糖尿病病例报告。仅在RFX2基因中变异c.1894G>A的患者中，没有一个家庭成员受到先证者变体的影响。
结论：体内功能β细胞研究允许选择五个最可能的单基因糖尿病基因。家族共分离分析表明，RFX2基因的新变异可能是糖尿病的一个可能原因。未来的体外功能分析是必要的，以支持或排除作为糖尿病原因的遗传背景。
方法：这是“立陶宛遗传性糖尿病”项目的延续，该项目包括1209名糖尿病患者。在88.1%（n=1065）的患者中，自身免疫标志物的先前筛查证实了1型糖尿病（T1D）的诊断，靶向下一代测序在MODY基因中鉴定出3.5%(n=42)的致病变异。随后，102例患者被归类为病因不明的糖尿病。12/102被发现在潜在的糖尿病基因中具有新的变异（RFX2，RREB1，SLC5A1（3名患者在该基因中具有变异），GCKR,MC4R,CASP10,TMPRSS6,HGFAC,DACH1,ZBED3)。进行共分离分析和MMTT以研究具有特定变体的受试者中的β细胞功能。
结果：MMTT分析表明，在MC4R中具有变体的先证者，CASP10,TMPRSS6,HGFAC,SLC5A1（c.1415T>C）具有足够的残留β细胞功能，刺激的C肽（CP）>200pmol/L。7个个体在RFX2，RREB1，GCKR，DACH1，ZBED3和SLC5A1（c.1415T>C，和c.932A>T）表现为完全β细胞衰竭。当比较糖尿病发作时的年龄和持续时间时，在具有足够CP产生的患者和完全β细胞衰竭的患者之间没有发现统计学差异。共隔离分析包括19名家庭成员；其中没有糖尿病病例报告。仅在RFX2基因中变异c.1894G>A的患者中，没有一个家庭成员受到先证者变体的影响。
结论：体内功能β细胞研究允许选择五个最可能的单基因糖尿病基因。家族共分离分析表明，RFX2基因的新变异可能是糖尿病的一个可能原因。未来的体外功能分析是必要的，以支持或排除作为糖尿病原因的遗传背景。
