Association of peripheral blood DNA methylation level with Alzheimer’s disease progression

Background: Identifying biomarkers associated with Alzheimer's disease (AD) progression may enable patient enrichment and improve clinical trial designs. Epigenome-wide association studies have revealed correlations between DNA methylation at cytosine-phosphate-guanine (CpG) sites and AD pathology and diagnosis. Here, we report relationships between peripheral blood DNA methylation profiles measured using Infinium® MethylationEPIC BeadChip and AD progression in participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. Results: The rate of cognitive decline from initial DNA sampling visit to subsequent visits was estimated by the slopes of the modified Preclinical Alzheimer Cognitive Composite (mPACC; mPACCdigit and mPACCtrailsB) and Clinical Dementia Rating Scale Sum of Boxes (CDR-SB) plots using robust linear regression in cognitively normal (CN) participants and patients with mild cognitive impairment (MCI), respectively. In addition, diagnosis conversion status was assessed using a dichotomized endpoint. Two CpG sites were significantly associated with the slope of mPACC in CN participants (P < 5.79 × 10-8 [Bonferroni correction threshold]); cg00386386 was associated with the slope of mPACCdigit, and cg09422696 annotated to RP11-661A12.5 was associated with the slope of CDR-SB. No significant CpG sites associated with diagnosis conversion status were identified. Genes involved in cognition and learning were enriched. A total of 19, 13, and 5 differentially methylated regions (DMRs) associated with the slopes of mPACCtrailsB, mPACCdigit, and CDR-SB, respectively, were identified by both comb-p and DMRcate algorithms; these included DMRs annotated to HOXA4. Furthermore, 5 and 19 DMRs were associated with conversion status in CN and MCI participants, respectively. The most significant DMR was annotated to the AD-associated gene PM20D1 (chr1: 205,818,956 to 205,820,014 [13 probes], Sidak-corrected P = 7.74 × 10-24), which was associated with both the slope of CDR-SB and the MCI conversion status. Conclusion: Candidate CpG sites and regions in peripheral blood were identified as associated with the rate of cognitive decline in participants in the ADNI cohort. While we did not identify a single CpG site with sufficient clinical utility to be used by itself due to the observed effect size, a biosignature composed of DNA methylation changes may have utility as a prognostic biomarker for AD progression

Harnessing peripheral DNA methylation differences in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) to reveal novel biomarkers of disease

Background Alzheimer’s disease (AD) is a chronic progressive neurodegenerative disease impacting an estimated 44 million adults worldwide. The causal pathology of AD (accumulation of amyloid-beta and tau), precedes hallmark symptoms of dementia by more than a decade, necessitating development of early diagnostic markers of disease onset, particularly for new drugs that aim to modify disease processes. To evaluate differentially methylated positions (DMPs) as novel blood-based biomarkers of AD, we used a subset of 653 individuals with peripheral blood (PB) samples in the Alzheimer’s disease Neuroimaging Initiative (ADNI) consortium. The selected cohort of AD, mild cognitive impairment (MCI), and age-matched healthy controls (CN) all had imaging, genetics, transcriptomics, cerebrospinal protein markers, and comprehensive clinical records, providing a rich resource of concurrent multi-omics and phenotypic information on a well-phenotyped subset of ADNI participants. Results In this manuscript, we report cross-diagnosis differential peripheral DNA methylation in a cohort of AD, MCI, and age-matched CN individuals with longitudinal DNA methylation measurements. Epigenome-wide association studies (EWAS) were performed using a mixed model with repeated measures over time with a P value cutoff of 1 × 10−5 to test contrasts of pairwise differential peripheral methylation in AD vs CN, AD vs MCI, and MCI vs CN. The most highly significant differentially methylated loci also tracked with Mini Mental State Examination (MMSE) scores. Differentially methylated loci were enriched near brain and neurodegeneration-related genes (e.g., BDNF, BIN1, APOC1) validated using the genotype tissue expression project portal (GTex). Conclusions Our work shows that peripheral differential methylation between age-matched subjects with AD relative to healthy controls will provide opportunities to further investigate and validate differential methylation as a surrogate of disease. Given the inaccessibility of brain tissue, the PB-associated methylation marks may help identify the stage of disease and progression phenotype, information that would be central to bringing forward successful drugs for AD

Identification of HLA-DRB1 association to adalimumab immunogenicity.

Anti-drug antibody formation occurs with most biological agents across disease states, but the mechanism by which they are formed is unknown. The formation of anti-drug antibodies to adalimumab (AAA) may decrease its therapeutic effects in some patients. HLA alleles have been reported to be associated with autoantibody formation against interferons and other TNF inhibitors, but not adalimumab. We analyzed samples from 634 subjects with either rheumatoid arthritis (RA) or hidradenitis suppurativa (HS): 37 subjects (17 RA and 20 HS) developed AAA (AAA+) during adalimumab treatment and 597 subjects (348 RA, 249 HS) did not develop AAA (AAA-) during the clinical trials. Using next-generation sequencing-based HLA typing, we identified three protective HLA alleles (HLA-DQB1*05, HLA-DRB1*01,and HLA-DRB1*07) that were less prevalent in AAA+ than AAA-subjects (ORs: 0.4, 0.25 and 0.28, respectively; and P values: 0.012, 0.012 and 0.018, respectively) and two risk HLA alleles (HLA-DRB1*03 and HLA-DRB1*011) that were more abundant in AAA+ than AAA-subjects (ORs: 2.52, and 2.64, respectively; and P values: 0.006 and 0.019). Similar to the finding of Billiet et al. who found that carriage of the HLA-DRB1*03 allele was more prevalent in those with anti-infliximab antibodies (OR = 3.6, p = 0.002, 95% CI: [1.5,8.6]).), we found HLA-DRB1*03 allele was also more prevalent in anti-adalimumab positive (OR = 2.52, p = 0.006, 95% CI: [1.37,4.63]). The results suggest that specific HLA alleles may play a key role in developing AAAs in RA and HS patients treated with adalimumab

Additional file 1: Table S1. of Potential mechanisms of resistance to venetoclax and strategies to circumvent it

PCR primers and conditions. Figure S1. MCL-1 protein expression in parental cell lines and venetoclax-resistant populations. MCL-1 protein expression was measured using an assay developed based on the Luminex technology [19]. In brief, MCL-1 capture antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was custom-conjugated to Luminex carboxyl beads (bead region 9) by Millipore (St. Charles, MO, USA). MCL-1 detection antibody (Santa Cruz Biotechnology Inc.) was also conjugated to biotin through a custom service provided by Millipore. Cells were lysed in MILLIPLEX MAP lysis buffer 1 (Millipore Cat. no. 43-040, Danvers, MA, USA) containing protease inhibitor cocktail (Sigma). Data are presented as median fluorescent intensity (MFI). Equivalent amounts of protein from whole cell lysates generated from parental cell lines and their venetoclax-resistant versions were assessed. The signal was read using a Luminex FlexMap 3D system (Luminex, Austin, TX). Asterisks denote p < 0.05. (PPTX 79 kb

Overview of adalimumab RA and HS studies for HLA typing.

<p>Overview of adalimumab RA and HS studies for HLA typing.</p

HLA typing results for AAA formation in HLA-DQB1 and HLA-DRB1 regions in extension study for both RA and HS subjects by logistic model test.

<p>HLA typing results for AAA formation in HLA-DQB1 and HLA-DRB1 regions in extension study for both RA and HS subjects by logistic model test.</p

Forest plot of AAA formation (auto-antibody to adalimumab) according to the different HLA-DQB and DRB alleles in HS and RA by different tests.

<p><b>(A)</b> HLA Class II DQB and DRB alleles effect of combined subjects in HS and RA by odds ratio and 95% confidence intervals (CIs) on AAA formation by Fisher’s Exact Test. <b>(B)</b> HLA Class II DQB and DRB alleles effect of combined subjects in HS and RA by odds ratio and 95% confidence intervals (CIs) on AAA formation by Fisher’s Exact Test.</p

Comparison the Allelic association of HLA-DRB1*03 between published anti-infliximab antibody formation in IBD patients and in our AAA formation in RA and HS patients.

<p>The Allelic Association of HLA-DRB1*03 to Anti-infliximab Formation(ATI) among 76 ATI + Subjects and 116 ATI–Subjects [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195325#pone.0195325.ref022" target="_blank">22</a>], and in Formation in RA and HS among 37 AAA + Subjects and 597 AAA- Subjects by Fisher’s Exact Test and Logistic Model Test.</p