Dried blood spot metabolomics reveals a metabolic fingerprint with diagnostic potential for Diamond Blackfan Anaemia

Abstract

Summary The diagnostic evaluation of Diamond Blackfan Anaemia (DBA), an inherited bone marrow failure syndrome characterised by erythroid hypoplasia, is challenging because of a broad phenotypic variability and the lack of functional screening tests. In this study, we explored the potential of untargeted metabolomics to diagnose DBA. In dried blood spot samples from 18 DBA patients and 40 healthy controls, a total of 1752 unique metabolite features were identified. This metabolic fingerprint was incorporated into a machine‐learning algorithm, and a binary classification model was constructed using a training set. The model showed high performance characteristics (average accuracy 91·9%), and correct prediction of class was observed for all controls (n = 12) and all but one patient (n = 4/5) from the validation or ‘test’ set (accuracy 94%). Importantly, in patients with congenital dyserythropoietic anaemia (CDA) – an erythroid disorder with overlapping features – we observed a distinct metabolic profile, indicating the disease specificity of the DBA fingerprint and underlining its diagnostic potential. Furthermore, when exploring phenotypic heterogeneity, DBA treatment subgroups yielded discrete differences in metabolic profiles, which could hold future potential in understanding therapy responses. Our data demonstrate that untargeted metabolomics in dried blood spots is a promising new diagnostic tool for DBA.

Red Blood Cell Metabolism in Pyruvate Kinase Deficient Patients

“…Despite altered glycolysis, ATP levels were surprisingly found to be higher in PKD patients in this cohort. The data is inconsistent with standard ATP measurements in the literature and previous studies on whole blood from dried blood spots ( Dooijeweert et al, 2021 ), despite the lack of iatrogenic interventions (i.e., recent transfusion events at the time of blood draws) in any of the subjects investigated here. The high degree of reticulocytosis that accompanies of these patients may have impacted measurements of carboxylic acids (also elevated in PKD samples from earlier dried blood spot studies; Dooijeweert et al, 2021 ) and ATP production through residual mitochondrial activity, despite the lack of significant differences in the levels of most carboxylates between the two groups with the exception of succinate.…”

“…The data is inconsistent with standard ATP measurements in the literature and previous studies on whole blood from dried blood spots ( Dooijeweert et al, 2021 ), despite the lack of iatrogenic interventions (i.e., recent transfusion events at the time of blood draws) in any of the subjects investigated here. The high degree of reticulocytosis that accompanies of these patients may have impacted measurements of carboxylic acids (also elevated in PKD samples from earlier dried blood spot studies; Dooijeweert et al, 2021 ) and ATP production through residual mitochondrial activity, despite the lack of significant differences in the levels of most carboxylates between the two groups with the exception of succinate. Accumulation of succinate in PKD patients is suggestive of a potential disruption in the activity of succinate dehydrogenase (SDH) at complex II in mitochondria in other cells than RBCs, as a function of PKD-induced anemia/hypoxia, similarly to what observed in the face of pathological hemorrhagic ( D’Alessandro et al, 2017 ) or ischemic ( Chouchani et al, 2014 ) hypoxia in previous studies.…”

“…In conclusion, in the present study, we provide the first overview of RBC metabolism in patients with PKD, expanding on previous reports on whole blood from dried blood spots (Dooijeweert et al, 2021). Though limited in scope (i.e., small cohort with a single genetic mutation R479H), the study addresses the need for basic science to investigate pathologies targeting underrepresented minorities (four out of five PKD subjects were from Amish families), with the ultimate goal to target treatments to health disparities (Hunter et al, 2021).…”

“…While previous reports focused on dried blood spot analysis of whole blood from PKD patients ( Dooijeweert et al, 2021 ), in this study, we attempted to characterize the metabolome of RBCs from PKD patients. We hypothesized that PKD samples would show reprogramming of energy metabolism, with increases in early glycolytic metabolites and decreases in late glycolytic metabolites.…”

Interpretable machine learning methods for predictions in systems biology from omics data

“…Frontiers in Molecular Biosciences frontiersin.org Trainor et al, 2017;Alakwaa et al, 2018;Asakura et al, 2018;Date and Kikuchi, 2018;Hu et al, 2018;Stamate et al, 2019;Toubiana et al, 2019;Sha et al, 2021;van Dooijeweert et al, 2021) use a lower number of effective raw features for predictions than studies employing only transcriptomics (Sharma et al, 2019;Culley et al, 2020;Wang et al, 2020;Alghamdi et al, 2021;Wang et al, 2021). The two exceptions on the transcriptomics side (Culley et al, 2020;Alghamdi et al, 2021) originally had more raw features but some of them were omitted for at least one major analysis because some genes were not present in a metabolic network model.…”