LC–MS/MS-Based Absolute Quantitation of Hemoglobin Subunits from Dried Blood Spots Reveals Novel Biomarkers for α‑Thalassemia Silent Carriers

Identification of α-thalassemia silent carriers
is challenging
with conventional phenotype-based screening methods. A liquid chromatography
tandem mass spectrometry (LC–MS/MS)-based approach may offer
novel biomarkers to address this conundrum. In this study, we collected
dried blood spot samples from individuals with three α-thalassemia
subtypes for biomarker discovery and validation. We observed differential
expression patterns of hemoglobin subunits among various α-thalassemia
subtypes and normal controls through proteomic profiling of 51 samples
in the discovery phase. Then, we developed and optimized a multiple
reaction monitoring (MRM) assay to measure all detectable hemoglobin
subunits. The validation phase was conducted in a cohort of 462 samples.
Among the measured hemoglobin subunits, subunit μ was significantly
upregulated in all the α-thalassemia groups with distinct fold
changes. The hemoglobin subunit μ exhibits great potential as
a novel biomarker for α-thalassemia, especially for silent α-thalassemia.
We constructed predictive models based on the concentrations of hemoglobin
subunits and their ratios to classify the various subtypes of α-thalassemia.
In the binary classification problems of silent α-thalassemia
vs normal, non-deletional α-thalassemia vs normal, and deletional
α-thalassemia vs normal, the best performance of the models
achieved average ROCAUCs of 0.9505, 0.9430, and 0.9976 in the cross-validation,
respectively. In the multiclass model, the best performance achieved
an average ROCAUC of 0.9290 in cross-validation. The performance of
our MRM assay and models demonstrated that the hemoglobin subunit
μ would play a vital role in screening silent α-thalassemia
in clinical practice.