Discussion
The main strength of the whole project “Genetic Diabetes in Lithuania” is that it included the entire cohort of children (n=860) with diabetes and in the majority of young diabetic adults (n=349) in Lithuania, and it reflects our approach to the search for genetic diabetes cases. During this project, 3.5% (n=42) of subjects were identified to have MD caused by pathogenic variants in already known MD genes.7 Even after testing for pancreatic beta-cell autoimmune markers, and performing comprehensive targeted NGS, in 8.4% (102/1209) of the total cohort precise diabetes etiology was not identified. As expected, high-throughput sequencing brought several novel variants (11/102) in potential diabetes genes, in agreement with other authors reporting candidate gene variants in up to 10% of sequenced cohorts.26–28 Genomic individualized medicine not only brings the accurate molecular diagnosis and the most effective treatment plan for the patient but also delivers surveillance plan for family members; furthermore, it has proven cost-effectiveness for the healthcare system.29 Therefore, with this study, we also present our selection of potential genes for MD for further functional analysis in vitro.
A second strength of this study is that we chose CP measurements during MMTT rather than OGTT to assess beta-cell function, as MMTT is considered the gold model to evaluate endogenous insulin secretion, although it requires time and effort.22–25 Thus, the functional trial of beta cells in vivo during MMTT in our study showed that even after more than 2.5 years of diabetes duration, patients with novel variants in MC4R, CASP10, TMPRSS6, HGFAC, and SLC5A1 (one of brothers with c.1415T>C) still had sufficient residual pancreatic function, suggesting either slower beta-cell destruction or stable functional residual beta-cell mass. Furthermore, the rest of the subjects, who had variants in RFX2, RREB1, GCKR, DACH1, and ZBED3, also in SLC5A1, showed complete beta-cell failure and endogenous insulin deficiency. We believe that hyperglycemia correction with fast-acting insulin 2 hours prior to MMTT in subject with DACH1 gene variant could influence glycemia levels until the 90th minute, considering C-peptide levels.
Considering that several previous studies have shown that serum CP, fasting or random, and urinary CP/creatinine ratio are highly sensitive and specific biomarkers for discriminating T1D and other forms of diabetes, especially monogenic,30–32 we believe that our results suggest that patients with sufficient endogenous insulin production may be eligible for treatment optimization in the future, given that our previous treatment optimization trial in patients with maturity-onset diabetes of the young (MODY) showed the threshold of stimulated CP ≥332.5 pmol/L for a successful treatment transition (from insulin injections to oral therapy).21
Additional strength of our study is familial co-segregation analysis. This analysis linked the novel variants in candidate genes with diabetes phenotype. It revealed that the variant c.1894G>A in RFX2 gene could be a suitable candidate for functional analysis in vitro, as neither of healthy family members possessed the novel variant of the proband. Despite the positivity for ZnT8A, this proband was intentionally selected for NGS because of clinical suspicion of MD and data reported by other authors that up to 1–2% of patients with MODY are found to have positive autoimmune markers.33 Further genetic analysis revealed that the patient had a novel variant. RFX2 gene belongs to the regulatory factor binding to the X-box (RFX) transcription factors’ family.13 This family also includes RFX6 gene, which is a known beta-cell transcription factor and causes permanent neonatal diabetes, or MODY.2 3 Usually, pathogenic mutation in RFX6 presents with hypoplasia of pancreas and additional syndromic features, although none of them were reported in our patient. Moreover, the recent study in animal models showed that alterations in the expression of RFX2 gene may lead to increased susceptibility for autoimmune insulitis in beta cells.34 Taking into account all data, we believe that RFX2 could be a good candidate for functional analysis in cell cultures.
The main limitation of this study was the drop-out of patients due to cumbersome and time-consuming investigations. Furthermore, COVID-19 pandemic prolonged the recruitment of study subjects for a couple of years. It is reported by several authors that the willingness to participate in clinical trials during COVID-19 pandemic decreased up to 10–20%; some were closed because of insufficient sample sizes.35 36 Nevertheless, further investigations of the remaining families of our cohort are to be implemented. An additional limitation of our study is that we have not yet been able to reclassify novel genetic variants; therefore, we need future functional experiments of candidate genes in vitro.
In summary, functional beta-cell study in vivo allowed to select five most probable diabetes genes (MC4R, CASP10, TMPRSS6, HGFAC, SLC5A1) after proven endogenous insulin secretion during MMTT. Changes in RFX2 gene may have either a causative or predisposing role in diabetes pathophysiology. Further research directions with functional analysis in cell cultures of these genes could support or rule out the genetic background as a reason of diabetes.