Assembly of 809 whole mitochondrial genomes with clinical, imaging, and fluid biomarker phenotyping.

INTRODUCTION: Mitochondrial genetics are an important but largely neglected area of research in Alzheimer's disease. A major impediment is the lack of data sets. METHODS: We used an innovative, rigorous approach, combining several existing tools with our own, to accurately assemble and call variants in 809 whole mitochondrial genomes. RESULTS: To help address this impediment, we prepared a data set that consists of 809 complete and annotated mitochondrial genomes with samples from the Alzheimer's Disease Neuroimaging Initiative. These whole mitochondrial genomes include rich phenotyping, such as clinical, fluid biomarker, and imaging data, all of which is available through the Alzheimer's Disease Neuroimaging Initiative website. Genomes are cleaned, annotated, and prepared for analysis. DISCUSSION: These data provide an important resource for investigating the impact of mitochondrial genetic variation on risk for Alzheimer's disease and other phenotypes that have been measured in the Alzheimer's Disease Neuroimaging Initiative samples.

Genome-wide association study of CSF levels of 59 alzheimer's disease candidate proteins: significant associations with proteins involved in amyloid processing and inflammation.

Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aß42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10-10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.

Evaluating the necessity of PCR duplicate removal from next-generation sequencing data and a comparison of approaches.

BACKGROUND: Analyzing next-generation sequencing data is difficult because datasets are large, second generation sequencing platforms have high error rates, and because each position in the target genome (exome, transcriptome, etc.) is sequenced multiple times. Given these challenges, numerous bioinformatic algorithms have been developed to analyze these data. These algorithms aim to find an appropriate balance between data loss, errors, analysis time, and memory footprint. Typical analysis pipelines require multiple steps. If one or more of these steps is unnecessary, it would significantly decrease compute time and data manipulation to remove the step. One step in many pipelines is PCR duplicate removal, where PCR duplicates arise from multiple PCR products from the same template molecule binding on the flowcell. These are often removed because there is concern they can lead to false positive variant calls. Picard (MarkDuplicates) and SAMTools (rmdup) are the two main softwares used for PCR duplicate removal. RESULTS: Approximately 92 % of the 17+ million variants called were called whether we removed duplicates with Picard or SAMTools, or left the PCR duplicates in the dataset. There were no significant differences between the unique variant sets when comparing the transition/transversion ratios (p = 1.0), percentage of novel variants (p = 0.99), average population frequencies (p = 0.99), and the percentage of protein-changing variants (p = 1.0). Results were similar for variants in the American College of Medical Genetics genes. Genotype concordance between NGS and SNP chips was above 99 % for all genotype groups (e.g., homozygous reference). CONCLUSIONS: Our results suggest that PCR duplicate removal has minimal effect on the accuracy of subsequent variant calls.

Interaction between variants in CLU and MS4A4E modulates Alzheimer's disease risk.

INTRODUCTION: Ebbert et al. reported gene-gene interactions between rs11136000-rs670139 (CLU-MS4A4E) and rs3865444-rs670139 (CD33-MS4A4E). We evaluate these interactions in the largest data set for an epistasis study. METHODS: We tested interactions using 3837 cases and 4145 controls from Alzheimer's Disease Genetics Consortium using meta-analyses and permutation analyses. We repeated meta-analyses stratified by apolipoprotein E (APOE) ε4 status, estimated combined odds ratio (OR) and population attributable fraction (cPAF), and explored causal variants. RESULTS: Results support the CLU-MS4A4E interaction and a dominant effect. An association between CLU-MS4A4E and APOE ε4 negative status exists. The estimated synergy factor, OR, and cPAF for rs11136000-rs670139 are 2.23, 2.45, and 8.0, respectively. We identified potential causal variants. DISCUSSION: We replicated the CLU-MS4A4E interaction in a large case-control series and observed APOE ε4 and possible dominant effect. The CLU-MS4A4E OR is higher than any Alzheimer's disease locus except APOE ε4, APP, and TREM2. We estimated an 8% decrease in Alzheimer's disease incidence without CLU-MS4A4E risk alleles and identified potential causal variants.

Variant Tool Chest: an improved tool to analyze and manipulate variant call format (VCF) files.

BACKGROUND: Since the advent of next-generation sequencing many previously untestable hypotheses have been realized. Next-generation sequencing has been used for a wide range of studies in diverse fields such as population and medical genetics, phylogenetics, microbiology, and others. However, this novel technology has created unanticipated challenges such as the large numbers of genetic variants. Each caucasian genome has more than four million single nucleotide variants, insertions and deletions, copy number variants, and structural variants. Several formats have been suggested for storing these variants; however, the variant call format (VCF) has become the community standard. RESULTS: We developed new software called the Variant Tool Chest (VTC) to provide much needed tools to work with VCF files. VTC provides a variety of tools for manipulating, comparing, and analyzing VCF files beyond the functionality of existing tools. In addition, VTC was written to be easily extended with new tools. CONCLUSIONS: Variant Tool Chest brings new and important functionality that complements and integrates well with existing software. VTC is available at https://github.com/mebbert/VariantToolChest.

Surveying the landscape of RNA isoform diversity and expression across 9 GTEx tissues using long-read sequencing data.

Even though alternative RNA splicing was discovered in 1977 (nearly 50 years ago), we still understand very little about most isoforms arising from a single gene, including in which tissues they are expressed and if their functions differ. Human gene annotations suggest remarkable transcriptional complexity, with approximately 252,798 distinct RNA isoform annotations from 62,710 gene bodies (Ensembl v109; 2023), emphasizing the need to understand their biological effects. For example, 256 gene bodies have ≥50 annotated isoforms and 30 have ≥100, where one protein-coding gene (MAPK10) even has 192 distinct RNA isoform annotations. Whether such isoform diversity results from biological noise (i.e., spurious alternative splicing) or whether it represents biological intent and specialized functions (even if subtle) remains a mystery. Recent studies by Aguzzoli-Heberle et al., Leung et al., and Glinos et al. demonstrate long-read RNAseq enables improved RNA isoform quantification for essentially any tissue, cell type, or biological condition (e.g., disease, development, aging, etc.) making it possible to better assess individual isoform expression and function. While each study provided important discoveries related to RNA isoform diversity, deeper exploration is needed. We sought, in part, to quantify real isoform usage across tissues (compared to annotations) and explore whether observed diversity is biological noise or intent. We used long-read RNAseq data from 58 GTEx samples across nine tissues (three brain, two heart, muscle, lung, liver, and cultured fibroblasts) generated by Glinos et al. and found considerable isoform diversity within and across tissues. Cerebellar hemisphere was the most transcriptionally complex tissue (22,522 distinct isoforms; 3,726 unique); liver was least diverse (12,435 isoforms; 1,039 unique). We highlight gene clusters exhibiting high tissue-specific isoform diversity per tissue (e.g., TPM1 expresses 19 in heart's atrial appendage), and specific genes (PAX6 and TPM1) that counterintuitively exhibit evidence that their expressed isoform diversity results from both biological noise and intent. We also validated 447 of the 700 new isoforms discovered by Aguzzoli-Heberle et al. and found that 88 were expressed in all nine tissues, while 58 were specific to a single tissue. This study represents a broad survey of the RNA isoform landscape, demonstrating isoform diversity across nine tissues and emphasizes the need to better understand how individual isoforms from a single gene body contribute to human health and disease.

Mapping medically relevant RNA isoform diversity in the aged human frontal cortex with deep long-read RNA-seq.

Determining whether the RNA isoforms from medically relevant genes have distinct functions could facilitate direct targeting of RNA isoforms for disease treatment. Here, as a step toward this goal for neurological diseases, we sequenced 12 postmortem, aged human frontal cortices (6 Alzheimer disease cases and 6 controls; 50% female) using one Oxford Nanopore PromethION flow cell per sample. We identified 1,917 medically relevant genes expressing multiple isoforms in the frontal cortex where 1,018 had multiple isoforms with different protein-coding sequences. Of these 1,018 genes, 57 are implicated in brain-related diseases including major depression, schizophrenia, Parkinson's disease and Alzheimer disease. Our study also uncovered 53 new RNA isoforms in medically relevant genes, including several where the new isoform was one of the most highly expressed for that gene. We also reported on five mitochondrially encoded, spliced RNA isoforms. We found 99 differentially expressed RNA isoforms between cases with Alzheimer disease and controls.

Using deep long-read RNAseq in Alzheimer's disease brain to assess medical relevance of RNA isoform diversity.

Due to alternative splicing, human protein-coding genes average over eight RNA isoforms, resulting in nearly four distinct protein coding sequences per gene. Long-read RNAseq (IsoSeq) enables more accurate quantification of isoforms, shedding light on their specific roles. To assess the medical relevance of measuring RNA isoform expression, we sequenced 12 aged human frontal cortices (6 Alzheimer's disease cases and 6 controls; 50% female) using one Oxford Nanopore PromethION flow cell per sample. Our study uncovered 53 new high-confidence RNA isoforms in medically relevant genes, including several where the new isoform was one of the most highly expressed for that gene. Specific examples include WDR4 (61%; microcephaly), MYL3 (44%; hypertrophic cardiomyopathy), and MTHFS (25%; major depression, schizophrenia, bipolar disorder). Other notable genes with new high-confidence isoforms include CPLX2 (10%; schizophrenia, epilepsy) and MAOB (9%; targeted for Parkinson's disease treatment). We identified 1,917 medically relevant genes expressing multiple isoforms in human frontal cortex, where 1,018 had multiple isoforms with different protein coding sequences, demonstrating the need to better understand how individual isoforms from a single gene body are involved in human health and disease, if at all. Exactly 98 of the 1,917 genes are implicated in brain-related diseases, including Alzheimer's disease genes such as APP (Aß precursor protein; five), MAPT (tau protein; four), and BIN1 (eight). As proof of concept, we also found 99 differentially expressed RNA isoforms between Alzheimer's cases and controls, despite the genes themselves not exhibiting differential expression. Our findings highlight the significant knowledge gaps in RNA isoform diversity and their medical relevance. Deep long-read RNA sequencing will be necessary going forward to fully comprehend the medical relevance of individual isoforms for a "single" gene.

A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.