Determining APOE ε4 carriership by NUcleic acid Linked Immuno‐Sandwich Assay (NULISA)

Abstract Background The APOE ε4 variant is the largest known genetic risk factor for late‐onset sporadic Alzheimer's disease (AD). Recent blood biomarker models include APOE ε4 status with plasma p‐tau217 for higher accuracy for AD pathology. Thus, protein assays that can accurately determine ε4 carriership simultaneously with plasma p‐tau217 would be advantageous for clinical use. This study aims to evaluate the concordance between the NULISA ApoE4 protein assay and conventional genetic testing for APOE measurement. Method We included two independent cohorts; cohort 1 (Translational Biomarker for Aging and Dementia Cohort [TRIAD]) consisted of 341 participants (mean [SD] age, 64.8[16.1] years; 213 females[62.4%]) and cohort 2 consisted of 38 participants (72.1[16.1] years; 21 females[55.2%]). APOE genotyping was determined by the TaqMan® SNP Genotyping Assay. In cohort 1, ApoE4 and p‐tau217 levels (NPQ) were quantified by the NULISAseq CNS disease panel from Alamar Biosciences. In cohort 2, ApoE4% (ApoE4/ApoE total ) were quantified by a prototype singleplex assay from Alamar Biosciences Result In cohort 1, we included 341 participants with APOE genotyping (ε4 non‐carriers=221; ε4 carriers=120). ApoE4 NPQ values correctly identified 88.3% of carriers and 94.1% of non‐carriers (Figure 1A). Homozygous ε2 and ε4 carriers were entirely identified. Higher ApoE4 NPQ levels were observed in ε4/ε4 carriers (median [SD], 16.4[0.52]) compared to ε3/ε4 carriers (12.3[5.11]; P<0.0001) but an overlap remained. In these participants, the simultaneously measurement of NULISA plasma p‐tau217 was significantly increased in Aβ+ participants compared to Aβ‐ participants (AUC=0.941; P<0.0001). In cohort 2, we included 38 participants with APOE genotyping (ε3/ε3, n=26; ε3/ε4, n=9; ε4/ε4, n=6) and calculated the ApoE4% from a prototype singleplex NULISA method. Here, ε3/ε3 (0.03 [1.43]), ε3/ε4 (9.37 [1.61] and ε4/ε4 (41.9 [18.0]) individuals were classified with 100% accuracy (Figure 1B). Conclusion Our study assessed ApoE protein assays to determine APOE genotype. The ApoE4 assay within the NULISAseq CNS panel demonstrated high accuracy in distinguishing APOE ε4 carriers from non‐carriers, with some discordance. The reason for the discordance subject to further studies. However, ApoE4% quantification using a prototype singleplex assay distinguished ε4 homozygous, ε4 heterozygous, and non‐carriers with 100% accuracy. This pilot study demonstrates the ability to concurrently determine APOE status and p‐tau217 levels for more accurate diagnostic models of AD pathology.