Integrative Population Analysis of MICA and MICB Using Unsupervised Machine Learning in a Large Histocompatibility Laboratory Cohort

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Background: Non-classical MHC class I molecules MICA and MICB are stress-inducible NKG2D ligands that contribute to immune surveillance, non-HLA antibody formation, and alloreactivity in solid organ and hematopoietic stem cell transplantation; population-level data for Southern Europe remain limited. Methods: High-resolution MICA and MICB genotyping was performed in 1364 unrelated individuals from southern Portugal using a hybrid-capture next-generation sequencing workflow, and allele calls were analyzed with standard population-genetic metrics (allele and genotype frequencies, heterozygosity, Hardy–Weinsberg equilibrium, and LD-like D, D′, r2) and multilocus allele presence/absence encodings explored by k-means clustering, spectral clustering, principal component analysis, t-distributed stochastic neighbor embedding, and uniform manifold approximation and projection. Results: Forty-two MICA and twenty-two MICB alleles were identified; MICA*002:01, MICA*004:01, MICA*008:01, MICA*008:04 and MICB*002:01, MICB*004:01, MICB*005:02, MICB*008:01 were most frequent, and most individuals carried at least two distinct MICA and two distinct MICB allotypes. Co-occurrence and LD-like analyses revealed conserved MICA–MICB combinations, including a strong association between MICA*009:02 and MICB*005:06, while unsupervised analyses identified partially overlapping multilocus genotype backgrounds and recurrent four-allele constellations. Conclusion: These findings provide a detailed non-classical MHC reference for southern Portugal and a multilocus framework to support interpretation of non-HLA antibodies and MICA/MICB-aware donor evaluation in selected clinical scenarios, as well as the development of machine learning-based immunologic risk models.