The prevalence and incidence of diabetes mellitus (DM) is increasing due to lifestyle shifts toward western-type diets and sedentary behaviors. There is, however, considerable heterogeneity in the association of dietary intake with the risk for developing type 2 diabetes mellitus (T2DM) and glycemic traits, which has been attributed to human genetic variation. Indeed, studies in twins have indicated a major contribution of genetics to the development of T2DM, which is comparable to the impact of diet (1). Genome-wide association studies (GWAS) have identified more than 140 genomic loci linked to increased T2DM predisposition (2) and individual meal responses (3). Several of these variants have been explored in combination to formulate genetic risk scores (GRS) to better predict the relative risk of T2DM development (4).

Medical nutrition therapy (MNT) is endorsed by both the Academy of Nutrition and Dietetics and the American Diabetes Association (ADA) for the management of adults with T2DM or pre-diabetes toward achieving euglycemia (5, 6). However, specific recommendations for the proportion of macronutrients, or oral nutrient supplementation (ONS) guidelines for the MNT of T2DM are lacking (6). As an example, the Obesity Society recommends a carbohydrate (CHO) intake between 45–65% of the energy intake (EI), 15–35% from protein, and 30% from total fat [10% from polyunsaturated fatty acid (PUFA), 15–20% from monounsaturated fatty acids (MUFA), 7% from saturated fatty acids (SFA)] for the management of T2DM (7). On the other hand, the Mediterranean diet, which confers significant benefits to T2DM prevention, is considered a high-fat diet (>40% of the EI from total fat), rich in MUFA but poor in SFA (8–10). This ambiguity in the MNT recommendations for patients with T2DM likely reflects the need to provide dietary and lifestyle advice personalized to the patient's bioclinical, phenotypic, lifestyle, and genetic data, thus maximizing its impact (11).

Several lines of clinical and experimental evidence call for a major conceptual and practical shift in dietetics toward the provision of precision nutrition care, parallel to the progress in precision medicine. Genetic variation is an important source of nutrition-related metabolic heterogeneity (12). Thus far, many observational studies have reported associations between specific genetic variants, predominantly single nucleotide polymorphisms (SNPs), and the health outcomes to the provision of specific macro- and micro-nutrients and ONS, including T2DM (11, 13, 14). However, how this information could be combined to provide dietary and lifestyle recommendations in T2DM remains poorly explored.

Since Hogben and Sim first conceived n-of-1 trials as components of pragmatic clinical practice in the 1950s (15), their application has gained residence. Today, the n-of-1 trial design represents the cornerstone of precision medicine and nutrition, offering insight on the individual patient response without requiring post-hoc analyses (16–19). The implementation of n-of-1 trials is used to compare interventions and find the most effective one for each patient, increasing treatment-response, reducing polypharmacy, minimizing adverse events, while conserving health care resources (20).

In recent years, nutrigenetic investigations have aided the delivery of MNT interventions in large-scale dietary trials, with the PREDIMED, the Look AHEAD and the POUNDS Lost clinical trials being notable examples. These trials reported milestone findings for the association of single SNPs with the outcome of MNT, including the interaction of a TCF7L2 variant with a Mediterranean diet (MD) pattern for glycemic traits (21) and the interaction of a MTNR1b variant with dietary fat intake for glycemic traits (22–24). They have also identified associations between GRS for diabetes, and protein intake influencing insulin resistance (25). However, despite promising results, research and validations in different populations are still required. The conduction of nutritional n-of-1 trials may represent an advantageous method for gene-nutrition and diabetes-nutrition related research. Determination of the optimal personalized nutritional strategies, based on such trials is an essential step toward the delivery of precision prevention in the treatment of diabetes in the near future.

The present case series of n-of-1 cross-over superiority trials aimed to assess the effectiveness of precision, genetically guided MNT interventions vs. conventional MNT, in patients with pre-diabetes or T2DM.

The horizontal approach to the provision of dietary recommendations has been questioned (52) as it often fails to address particularities at the individual patient level. Approximately 70 years ago, Williams (53) was the first to envisage personalized nutritional recommendations that take into account “genetic patterns” and suggested that patients are “far from standardized specimens.” Although this concept remained dormant for several decades, the potential of precision nutrition in health and disease management is nowadays increasingly recognized (11, 13, 14, 54) and corroborated by several observational studies linking dietary outcomes to genetic variants (55–59). Along these lines, results from the Diabetes Prevention Program have suggested that, although a standardized intensive lifestyle intervention reduces DM risk (60), its efficacy varies across individuals (61), and is determined by genetic factors (62).

Conceptually, the series of n-of-1 trials presented herein addresses the reverse. It explores combinations of genetic variants associated with responses to dietary and lifestyle parameters to provide personalized dietary plans for patients with T2DM or pre-diabetes. We applied a quasi-experimental n-of-1 trials design as the most appropriate to address the effectiveness of conventional vs. genotype-based MNT treatment in the same patient. This methodology has been successfully explored in patient-centered medicine and nutrition studies (18, 63, 64), including DM (65), and can form a pragmatic research extension of the standard care.

The results provide proof-of-principle that we can achieve better glycaemic control in T2DM patients by coupling MNT to genetic information as a predictor of optimal macronutrient composition and ONS in a personalized manner. Both patients with pre-diabetes experienced remission of the condition. We particularly note that the successful outcome of these interventions entailed very different macronutrient compositions, ranging from a high-fat traditional Mediterranean dietary pattern to a diet with greater-protein intake. We thus propose that genetically-guided MNT may in fact aid in the resolution of the “ambiguity” regarding the ideal macronutrient composition for the management of DM (6–8). Furthermore, opting for genetically-informed dietary recommendations may enhance patient adherence. For example, informing the second patient of his genetic susceptibility to increased risk for T2DM at high BCAA intake led to voluntary changes in lifestyle choices toward abandoning BCAA ONS that he was unwilling to do otherwise. Thus, adherence to genetically-guided lifestyle recommendations is likely to amplify health outcomes. Indeed, several surveys conclude that individuals are more likely to adhere to dietary recommendations if these are based on their genetic profile (66, 67), while, on the other hand, improving adherence to healthy dietary patterns could attenuate the genetic predisposition for weight gain (57).

Despite the complexity of gene-nutrient interactions, genetic (GRS) or polygenic risk scores (PRS) coupled with mathematical predictive models and machine-learning algorithms are expected to accelerate the translation of genetic information to nutritional recommendations (68) and to have a major impact on the practical applications of nutrigenetics to precision nutrition (13). Results presented herein support this notion and confirm the utility of GRS in guiding nutritional recommendations in T2DM. It could be argued that the GRS used herein have not been adjusted for ancestry and this may indeed be a limitation of our study. Nevertheless, our results confirm the utility of GRS in guiding nutritional recommendations in T2DM in Greek similar to other populations (25, 46).

Other limitations of the present study include the possible carryover effect of the first intervention cycle which was addressed by testing for treatment-by-period interaction, as suggested for nutrition clinical trials (69). We note that a rule of thumb for the ideal duration of the wash-out period in dietary interventions does not exist, with the exception of ONS trials (70). Nevertheless, according to Senn (71), performing analyses for assessing the carryover effect of cross-over trials is not advised and analyzing data according to treatment-by-period interaction is the preferred strategy. In this manner, the Cochrane handbook suggests that although long wash-out periods can avoid the carryover effect, this is not deemed essential (72). Wash-out periods should also take into consideration ethical aspects related to maintenance of MNT in T2DM patients, including the violation of the equipoise principle (73–75). Of note, the importance of a wash-out period separating treatment phases in n-of-1 trials has also been debated (76). According to the DEcIDE methods center N-of-1 guidance Panel (20), often, the benefits of an intervention wash-out quickly, but the risks of adverse events persist.

An additional limitation of the present series may involve the small number of participants. However, the n-of-1 design is unanimously the most appropriate one for the implementation of precision medicine/nutrition interventions in the pragmatic setting (77, 78). As per Mizra and Guyatt (79), for the design to be suitable, the disease course must involve a chronic and relatively stable condition, such as in T2DM. A possible bottleneck in this design may involve the higher probability for a type-I error, however, aggregation of n-of-1 trials appears to outperform traditional RCTs in many methodological domains (80). According to Kravitz et al. (81), the importance of precision medicine stems from the individual heterogeneity in the treatment effect, thus n-of-1 trials represent the most appropriate method to gather information regarding the comparative effectiveness and adverse events of a novel therapy as compared to the conventional treatment, in order to inform pragmatic treatment decisions. In this regard, n-of-1 trials retain promise for the advancement of precision medicine (81) and precision nutrition (14, 63). We argue that the only caveat in relation to the small number of participants included herein might stem from the high individual heterogeneity and the subsequent inability to pool findings to perform group statistical analyses with respect to the two intervention cycles.

Regarding the lack of repetition of the AB intervention phases, the AB quasi-experimental design is considered as the most appropriate in defining the most effective, adverse-event-free therapy for each patient (18, 82). On the other hand, repetitions of the AB phases in a sequential manner is to n-of-1 trials, what sample size is for parallel arm randomized controlled trials (20) and can defend against random error. Nevertheless, the simple quasi-experimental design employed herein allows for the direct comparison of treatments A (conventional MNT) and B (genotype-guided MNT) and protects against several forms of systematic error (including history, testing, regression to the mean) (20, 83).

Finally, an additional limitation involves the lack of recorded data regarding the patients' perspective with respect to the treatments. Future studies based on the proof-of-principle results presented herein will address these issues.

Through these limited in number, yet carefully designed trials and investigated cases, we have herein addressed a major gap in linking the observational nature of gene-diet interactions to an effective step-by-step process required for the implementation of precision nutrition interventions (14, 84). Supplementary Figure 2 outlines the strategy followed in the present case series, based on the nutrition care process (NCP) model that may serve as a guide for the implementation of innovative genotype-directed MNTs in T2DM as well as other polygenic, multifactorial pathologies. Overall, the present series of n-of-1 superiority trials indicates that precision nutrition is both feasible and effective in adults with pre-diabetes and T2DM. Given the worldwide prevalence of T2DM, we anticipate that this approach may assist nutritionists in providing more personalized nutritional recommendations, enabling patient adherence and improved health outcomes.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Bioethics Committee of the National and Kapodistrian University of Athens Medical School. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

KG and AE: conceptualization, methodology, and resources. KG: assays. EL and KG: data collection. KG, AE, and MG: data curation. DS, EL, and DG: writing—review and editing. AE: visualization and project administration. All authors have read and agreed to the published version of the manuscript. All authors contributed to the article and approved the submitted version.

Author KG was employed by Embiodiagnostics Biology Research Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.