Top Variants Research Articles

Multi-omics correlates of insulin resistance and circadian parameters mapped directly from human serum.

While it is generally known that metabolic disorders and circadian dysfunction are intertwined, how the two systems affect each other is not well understood, nor are the genetic factors that might exacerbate this pathological interaction. Blood chemistry is profoundly changed in metabolic disorders, and we have previously shown that serum factors change cellular clock properties. To investigate if circulating factors altered in metabolic disorders have circadian modifying effects, and whether these effects are of genetic origin, we measured circadian rhythms in U2OS cell in the presence of serum collected from diabetic, obese or control subjects. We observed that circadian period lengthening in U2OS cells was associated with serum chemistry that is characteristic of insulin resistance. Characterizing the genetic variants that altered circadian period length by genome-wide association analysis, we found that one of the top variants mapped to the E3 ubiquitin ligase MARCH1 involved in insulin sensitivity. Confirming our data, the serum circadian modifying variants were also enriched in type 2 diabetes and chronotype variants identified in the UK Biobank cohort. Finally, to identify serum factors that might be involved in period lengthening, we performed detailed metabolomics and found that the circadian modifying variants are particularly associated with branched chain amino acids, whose levels are known to correlate with diabetes and insulin resistance. Overall, our multi-omics data showed comprehensively that systemic factors serve as a path through which metabolic disorders influence circadian system, and these can be examined in human populations directly by simple cellular assays in common cultured cells.

High-throughput characterization of functional variants highlights heterogeneity and polygenicity underlying lung cancer susceptibility

Genome-wide association studies (GWASs) have identified numerous lung cancer risk-associated loci. However, decoding molecular mechanisms of these associations is challenging since most of these genetic variants are non-protein-coding with unknown function. Here, we implemented massively parallel reporter assays (MPRAs) to simultaneously measure the allelic transcriptional activity of risk-associated variants. We tested 2,245 variants at 42 loci from 3 recent GWASs in East Asian and European populations in the context of two major lung cancer histological types and exposure to benzo(a)pyrene. This MPRA approach identified one or more variants (median 11 variants) with significant effects on transcriptional activity at 88% of GWAS loci. Multimodal integration of lung-specific epigenomic data demonstrated that 63% of the loci harbored multiple potentially functional variants in linkage disequilibrium. While 22% of the significant variants showed allelic effects in both A549 (adenocarcinoma) and H520 (squamous cell carcinoma) cell lines, a subset of the functional variants displayed a significant cell-type interaction. Transcription factor analyses nominated potential regulators of the functional variants, including those with cell-type-specific expression and those predicted to bind multiple potentially functional variants across the GWAS loci. Linking functional variants to target genes based on four complementary approaches identified candidate susceptibility genes, including those affecting lung cancer cell growth. CRISPR interference of the top functional variant at 20q13.33 validated variant-to-gene connections, including RTEL1, SOX18, and ARFRP1. Our data provide a comprehensive functional analysis of lung cancer GWAS loci and help elucidate the molecular basis of heterogeneity and polygenicity underlying lung cancer susceptibility.

Genetic analyses of inflammatory polyneuropathy and chronic inflammatory demyelinating polyradiculoneuropathy identified candidate genes

Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare, immune-mediated disorder in which an aberrant immune response causes demyelination and axonal damage of the peripheral nerves. Genetic contribution to CIDP is unclear and no genome-wide association study (GWAS) has been reported so far. In this study, we aimed to identify CIDP-related risk loci, genes and pathways. We first focused on CIDP, and 516 CIDP cases and 403,545 controls were included in the GWAS analysis. We also investigated genetic risk for inflammatory polyneuropathy (IP), in which we performed a GWAS study using FinnGen data and combined the results with GWAS from UK biobank (UKBB) using a fixed-effect meta-analysis. A total of 1,261 IP cases and 823,730 controls were included in the analysis. Stratified analyses by gender were performed. Mendelian randomization (MR), colocalization, and transcriptome-wide association study (TWAS) analyses were performed to identify associated genes. Gene-set analyses were conducted to identify associated pathways. We identified one genome-wide significant locus at 20q13.33 for CIDP risk among women; the top variant located at the intron region of gene CDH4. Sex-combined MR, colocalization and TWAS analyses identified three candidate pathogenic genes for CIDP, five genes for IP. MAGMA gene-set analyses identified a total of 18 pathways related to IP or CIDP. Sex-stratified analyses identified three genes for IP among males; and two genes for IP among females. Our study identified suggestive risk genes and pathways for CIDP and IP. Functional analysis should be conducted to further confirm these associations.

Novel genome-wide significant germline genetic variants associated with venous thrombo-embolism (VTE) and arterial embolism (ATE) risk in patients with ALK and ROS1 fusion non-small cell lung cancer (NSCLC).

12092 Background: In a recent meta-analysis, non small cell lung cancers (NSCLC) with ALK or ROS1 fusions had higher rates of VTE compared to KRAS or EGFR mutant NSCLC, and were associated with increased ATE risk, particularly around diagnosis. The underlying mechanisms of how these somatic fusions affect thrombosis remain unclear. We examined clinico-demographic and germline genetic factors associated with VTE and ATE in NSCLC patients with ALK/ROS1 fusions in this first comprehensive genome wide association analysis (GWAS). Methods: In this prospective cohort, germline DNA from whole blood was obtained from 150 patients with ALK fusions and 32 with ROS1 fusions at Princess Margaret Cancer Centre (recruited 2014- 2023). Clinico-demographic, treatment and outcome data were collected from electronic medical charts. Overall survival (OS) by VTE or ATE status was assessed via Cox regression, treating ATE and VTE as time-varying covariates. Genotyping utilized the Infinium Global Screening Array (v3.0 Illumina); quality-control removed 3 patients from final analysis. Using linear regression, outcomes were weighted; no VTE/ATE events (0), one VTE/ATE event (1), or multiple ATE/VTEs (>2). Global significance was set at p < 5 x10-8 and adjusted for age, sex, body mass index (BMI), and ethnic/population stratification (top 3 principal components). Results: There were 97 females (54%) and 82 males (46%), mean age was 57.4 years, 70% had stage 4 disease, 96% adenocarcinoma; 35% experienced VTE/ATE at any time; 13% had 2+ VTE/ATE events. For those with VTE, 32 scored 1, 22 scored 2 and 6 scored 3 on Khorana score. Higher BMI was a significant risk factor for VTE/ATE (adjusted odds ratio 1.59 per 5-unit increase, p=0.01). Shorter OS was observed in patients with VTE (Hazard ratio (HR)=3.15, p<0.001) and ATE (HR=2.51, p=0.036), compared to those without. Two novel GWAS gene peaks on the Manhattan plot (previously not reported to be associated with VTE/ATE) had globally significant associations with risk of VTE/ATE: at Chromosome 6q15 (intergenic region between RNGTT and LOC101928936; 5/10 top variants with top risk-allele p=3.594E-09) and at 15p22 ( TLN2 gene; 2/10 top variants p=1.095E-08). TLN2 is a cytoskeletal protein involved in the assembly of actin filaments and linkages with extracellular matrices. Additional identified variants potentially associated with VTE/ATE in ALK/ROS1 fusion patients were found in: CTBP2, NALCN, WDR7, PAX7, ZNF385D, SORBS2, and CSMD1. Conclusions: Two novel globally significant variants, associated with VTE/ATE, are unique to patients with ALK/ROS1 fusion NSCLC. If validated, these biomarkers may help identify ALK/ROS1 patients who are at highest risk of VTE/ATE who may benefit from prophylactic anticoagulation. Further investigation and clinical evaluation are warranted.

SORBS1 expression as a biomarker for obesity and breast cancer prognosis: Insights from genomic analysis and survival outcomes.

e13050 Background: Obesity is a known risk factor for breast cancer development and poorer survival. Correlation between the expression of the adaptor protein SORBS1 (Sorbin and SH3 domain-containing protein 1) and body mass index (BMI). Methods: Bioinformatics analysis. Results: We analyzed published datasets and plotted SORBS1 expression against patient BMI. Our findings show a negative correlation between SORBS1 expression and BMI in both datasets (R = -0.42, p = 0.00087, and R = -0.45 p = 0.00083, respectively). We also observed a negative correlation between SORBS1 expression and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) (R = -0.43, p = 0.00016). Additionally, we examined BMI-associated variants of the SORBS1 gene in the Qatar Biobank cohort of 15k individuals. We identified a total of 26 variants that were associated with an elevated risk for BMI in this population at a nominal significance. The top risk variant associated with the BMI is a missense variant (rs35480352; beta=0.058, p-value = 3.3e-03) with a frequency of 9.1% for the risk allele in our population. These results suggest that SORBS1 expression decreases with obesity and diabetes. This is significant because obesity is a known risk factor for breast cancer development and poorer survival. We also examined the SORBS1 gene expression in breast adenocarcinoma using UCSC TOIL RNA-seq recompute and TCGA-BRCA genomic and methylation data. Out of the 963 patients analyzed, only 12 (1.2%) showed any SORBS1 gene alteration, such as mutation (0.7%), amplification (0.2%), or deep deletion (0.3%), indicating a low occurrence of genomic alterations. Furthermore, our findings revealed no significant difference in SORBS1 methylation levels between the tumor and matched normal samples, indicating that the SORBS1 gene is not hypermethylated in breast carcinoma. Interestingly, our comparison of SORBS1 expression values between tumor and normal samples indicated significant under-expression in BRCA (log2FC = -2.7, p = 1.07E-127). SORBS1 expression was found to vary across various clinical stages (p = 0.0082, Kruskal-Wallis rank test), exhibiting a decline in advanced cancer stages. Moreover, we detected a significant difference in SORBS1 expression among different molecular subtypes (p < 2E-16, Kruskal-Wallis rank test), with a lower expression level in more aggressive subtypes like HER2. Patients were classified into two groups according to their SORBS1 expression levels, using the median expression as a threshold: high and low. Conclusions: Our findings showed that the SORBS1 low group had substantially lower overall survival (log-rank p = 0.0024) and progression-free survival rates (log-rank p = 0.028). Silencing of SORBS1 in MCF-7 and MDA-MB-231 cells increased cell proliferation, migration, and invasion, underscoring its importance in breast cancer prognosis and as a target for intervention.

Pharmacogenomics of coronary artery response to intravenous gamma globulin in kawasaki disease

Kawasaki disease (KD) is a multisystem inflammatory illness of infants and young children that can result in acute vasculitis. The mechanism of coronary artery aneurysms (CAA) in KD despite intravenous gamma globulin (IVIG) treatment is not known. We performed a Whole Genome Sequencing (WGS) association analysis in a racially diverse cohort of KD patients treated with IVIG, both using AHA guidelines. We defined coronary aneurysm (CAA) (N = 234) as coronary z ≥ 2.5 and large coronary aneurysm (CAA/L) (N = 92) as z ≥ 5.0. We conducted logistic regression models to examine the association of genetic variants with CAA/L during acute KD and with persistence >6 weeks using an additive model between cases and 238 controls with no CAA. We adjusted for age, gender and three principal components of genetic ancestry. The top significant variants associated with CAA/L were in the intergenic regions (rs62154092 p < 6.32E–08 most significant). Variants in SMAT4, LOC100127, PTPRD, TCAF2 and KLRC2 were the most significant non-intergenic SNPs. Functional mapping and annotation (FUMA) analysis identified 12 genomic risk loci with eQTL or chromatin interactions mapped to 48 genes. Of these NDUFA5 has been implicated in KD CAA and MICU and ZMAT4 has potential functional implications. Genetic risk score using these 12 genomic risk loci yielded an area under the receiver operating characteristic curve (AUC) of 0.86. This pharmacogenomics study provides insights into the pathogenesis of CAA/L in IVIG-treated KD and shows that genomics can help define the cause of CAA/L to guide management and improve risk stratification of KD patients.

Investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment.

Defining genetic factors impacting chemotherapy failure can help to better predict response and identify drug resistance mechanisms. However, there is limited understanding of the contribution of inherited noncoding genetic variation on inter-individual differences in chemotherapy response in childhood acute lymphoblastic leukemia (ALL). Here we map inherited noncoding variants associated with treatment outcome and/or chemotherapeutic drug resistance to ALL cis-regulatory elements and investigate their gene regulatory potential and target gene connectivity using massively parallel reporter assays and three-dimensional chromatin looping assays, respectively. We identify 54 variants with transcriptional effects and high-confidence gene connectivity. Additionally, functional interrogation of the top variant, rs1247117, reveals changes in chromatin accessibility, PU.1 binding affinity and gene expression, and deletion of the genomic interval containing rs1247117 sensitizes cells to vincristine. Together, these data demonstrate that noncoding regulatory variants associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to antileukemic agents.

Abstract 2986: Germline signatures predicting toxicity and response to CTLA4 inhibitors

Abstract Background: Because germline microRNA-based variants predict toxicity risk to anti-PD-1/PDL-1 treatment, and CTLA4 inhibitors are often used in combination with them, we wanted to see if we could also predict toxicity and/or response to anti-CTLA4 therapy. Methods: The study included 69 patients from 3 institutions treated with anti-CTLA4 therapy alone. Patients were 23-89 years old with median age of 62. “Toxicity” was defined as grade 3 or higher toxicity and “Response” as a complete response, partial response, or stable disease. A panel of previously tested variants (n=139), age, sex and cycles of anti-CTLA4 were used to predict endpoints of interest. To balance model dimensionality we pre-screened variants and included them if they were marginally associated with our outcomes of interest via Fisher’s Exact Test or Jonckheere-Terpstra Test at a p-value threshold of &amp;lt; 0.2. We fit Elastic Net (EN), Random Forest (RF), and Boosted Tree (BT) models with up-sampling to predict outcomes of interest. Leave-one-out cross validation (LOOCV) was used to evaluate model performance, and the model with the highest LOOCV AUC (between all EN, RF, and BT) was chosen as the final model and re-fit using all observations. We also performed a stratified gene ontology (GO) analysis to assess biological differences between variants involved in the signatures. Our GO analysis compared gene clusters across groupings with an adjusted p-value cutoff of 0.05 against a universal genomic background. Results: 15 patients had toxicity and 21 patients had a response, with 27/139 and 25/139 variants marginally associated with these endpoints, respectively. Our toxicity model had a LOOCV AUC of 0.811 with the top variables being age, TNNT2_rs3729843, REV3L_rs465646, miR34b_c_rs4938723, and XRCC3_rs861539. Our response model had an LOOCV AUC of 0.716 with top variants being IL2RA_rs2476491, REV3L_rs465646, and IL10RB_rs2834167. Including anti-CTLA4 cycles improved the prediction accuracy for Response with an AUC 0.764. GO analysis revealed 117 pathways enriched in both the Toxicity and Response signatures, which were predominantly involved in pri-miRNA transcriptional regulation. Top GO terms in the Response signature (333 unique) were related to apoptosis and cell death. Top GO terms in the Toxicity signature (425 unique) were related to DNA replication and cellular signaling and response, including the interleukin-35-mediated signaling pathway. Conclusion: We identified germline signatures predicting toxicity and response following anti-CTLA4 therapy. These signatures differed from each other as well as from a previously identified signature of toxicity to anti-PD1/PDL-1 therapy, and GO analysis identified plausible underlying signaling pathways. Our findings continue to validate the potential of these variants to predict meaningful endpoints in cancer therapy, and thus further research in larger and more diverse cohorts is warranted. Citation Format: Joanne B. Weidhaas, Kristen McGreevy, Nicholas Marco, Nora Sundahl, Christopher R. Cabanski, Christine Spencer, Piet Ost, Donatello Telesca. Germline signatures predicting toxicity and response to CTLA4 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2986.

Abstract 2845: Novel genetic risk loci for B-cell acute lymphoblastic leukemia in African American children: Findings from the Admixture and Risk of Acute Leukemia (ADMIRAL) Study

Abstract Background: Racial and ethnic disparities in incidence and outcomes for childhood B-cell acute lymphoblastic leukemia (B-ALL) are well established. While genome-wide association studies (GWASs) among children of European (EUR) genetic ancestry have identified robust risk loci, comparable studies in children of African (AFR) genetic ancestry do not exist. Here we report on results from the first GWAS of B-ALL exclusive to AFR children. Methods: A total of 840 AFR cases (diagnosis age &amp;lt;21y) and 3360 sex-/ancestry-matched controls (from ADMIRAL and ZOE 2.0, a childhood caries study) were evaluated. We used 656 cases from the Children’s Oncology Group (COG) ALL frontline trials for discovery and 184 non-COG cases for replication. Samples were genotyped with the Illumina Global Diversity Array and imputed with the TOPMed reference panel. Ancestry was ascertained with RFMix-inferred global ancestry values and principal components analysis. Logistic regression models adjusted for cryptic population substructure evaluated variant risk associations. Discovery and replication results were meta-analyzed using an inverse variance-weighted fixed effects model. Results: We identified 7 genome-wide significant (GWS, P&amp;lt;5 × 10−8) variants, including 2 novel B-ALL risk loci with replication evidence (P&amp;lt;0.05) and striking per allele effect sizes: CNTN4 (meta-analysis odds ratio [OR]=2.09, P=1.4 × 10−11) and FAM174A (OR=2.94, P=1.2 × 10−15). Established B-ALL EUR risk loci were corroborated in these data (ARIDB5, IKZF1; P&amp;lt;5 × 10−8). Five additional novel loci were GWS after meta-analysis including at SLC37A4 (OR=2.02, P=1.1 × 10−8) and CBWD1 (OR=2.69, P=2.7 × 10−9), with suggestive associations in discovery data (P&amp;lt;5 × 10−6) and nominal replication (P&amp;lt;0.05). Notably, all novel risk alleles were low-frequency in reference AFR populations and AFR-specific (minor allele frequencies of 2-9% in 1000 Genomes AFR and &amp;lt;0.1% in non-AFR). Top variants had relevant in silico annotations; variants at CNTN4 and SLC37A4 had high predicted functional probability (&amp;gt;0.8, RegulomeDb). For example, annotations for a top CNTN4 signal suggest a potential leukemogenesis mechanism, overlapping a ChIP-seq peak for CEBPA in transdifferentiated B cells. In our replication data, a polygenic risk score (PRS) with 5 novel AFR-specific GWS variants from discovery had an appreciably stronger B-ALL risk association (top 25% vs rest: OR=2.03, P=1.2 × 10−4) compared to a published B-ALL PRS from a GWAS in EUR children (top 25% vs rest: OR=1.59, P=0.014). Conclusions: Despite the modest number of AFR B-ALL cases, we successfully detected and replicated genotype risk associations with considerable effect sizes. These results demonstrate the critical importance of increasing diversity in genomic studies of childhood ALL to promote discovery and reduce disparities. Citation Format: Cindy Im, Andrew Raduski, Lauren J. Mills, Rebecca A. Johnson, Andrew DeWan, Xiaomei Ma, Joseph L. Wiemels, Catherine Metayer, Jun J. Yang, Heather H. Nelson, Tianzhong Yang, Saonli Basu, Lucie M. Turcotte, Nathan Pankratz, Michael E. Scheurer, Logan G. Spector. Novel genetic risk loci for B-cell acute lymphoblastic leukemia in African American children: Findings from the Admixture and Risk of Acute Leukemia (ADMIRAL) Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2845.

Abstract 6139: Genetic modifiers of KRAS-mutant colorectal cancer

Abstract Somatic driver mutations in KRAS are found in ~40% of colorectal cancers (CRCs) and are associated with worse outcomes. In the US, individuals of African ancestry with CRC are more likely to have tumors with KRAS mutations than individuals of European ancestry, even after considering differences in socioeconomic status and other risk factors. Germline variants can influence somatic mutation events and may provide a selective advantage such that a mutated cell is more likely to progress to a cancer. Germline variants have been found to associate with somatic mutations in a variety of tumors. Based on ancestry differences in KRAS tumor mutation frequency, we hypothesized that germline variants altering molecular pathways that support KRAS tumorigenesis will associate with KRAS-mutant status. To test this, we performed a two-step association study using KRAS mutation status as our phenotype in individuals with microsatellite stable CRC. First, we conducted a discovery analysis using genome-wide germline genotypes and somatic mutation data from 6,386 non-Hispanic White CRC patients from the Cancer Genome Atlas and the GECCO consortium, considering sex and tumor location as covariates. To select variants for additional study, we used in-silico tools, such as expression quantitative trait loci (eQTL), to predict how associated variants may alter gene expression and chromatin. After reviewing relevant genomic features, excluding SNVs with minor allele frequency &amp;lt;10% and pruning SNVs in linkage disequilibrium (r2&amp;gt;0.75), we identified candidate loci for a multi-ancestry independent validation analysis. In the discovery analysis, no variants met genome-wide significance thresholds (p-value &amp;lt;5x10-8), though 3 SNVs associated with KRAS mutation status with p-values &amp;lt;1x10-6 and 50 with p-values &amp;lt;1x10-5. Of these 50 SNVs, 21 met criteria for validation. We chose 80 additional SNVs (p-value &amp;lt;1x10-4) with predicted functional effect, for a total of 101 SNVs for analyses. In our preliminary validation analysis of 1,487 individuals grouped by self-reported race, no variants had a significant association in all populations. The rs726800 variant showed nominal association with KRAS mutation status (p-value=0.028) in self-reported non-Hispanic White individuals. This variant is within potential cis-regulatory distance of HS3ST3A1 and COX10-DT, both of which have been reported as cancer biomarkers. Nominal associations were identified in self-reported Black (n=7 SNVs) and Hispanic or Latino (n=3 SNVs) individuals, though both groups had limited sample sizes. Additional validation samples are being analyzed, as well as analysis with adjustment for genetic ancestry. Future functional studies of top candidate variants will add mechanistic insight. In summary, we identified germline SNVs that associate with KRAS somatic mutations in CRC and may inform the cellular context that supports development of colorectal tumors with KRAS mutations. Citation Format: Nijole P. Tjader, Johnny Ramroop, Tanish Gandhi, Peter T. Campbell, Andrew T. Chan, Steven Gallinger, Graham G. Giles, Marc J. Gunter, Tabitha A. Harrison, Michael Hoffmeister, Polly A. Newcomb, Shuji Ogino, Amanda I. Phipps, Conghui Qu, Robert E. Schoen, Andrew Pellatt, Michael O. Woods, Bethany Van Guelpen, Patrick Stevens, Heather Hampel, Ulrike Peters, Joseph P. McElroy, Amanda E. Toland. Genetic modifiers of KRAS-mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6139.

No evidence for association between rs10191329 severity locus and longitudinal disease severity in 1813 relapse-onset multiple sclerosis patients from the MSBase registry

Background: The International Multiple Sclerosis Genetics Consortium and MultipleMS Consortium recently reported a genetic variant associated with multiple sclerosis (MS) severity. However, it remains unclear if these variants remain associated with more robust, longitudinal measures of disease severity. Methods: We examined the top variant, rs10191329, from Harroud et al.’s study in 1813 relapse-onset MS patients from the MSBase Registry to assess association with longitudinal disease severity. Results: Our analysis revealed no significant association between rs10191329 genotype and longitudinal binary disease severity (p > 0.05). Conclusion: These findings highlight the complexity of genetic factors mediating long-term MS outcomes and the need for further research.

Abstract P494: Gene-Level Gene-Environment Interaction Testing Reveals Rare Variants With Large Impacts on the Omega-3 Fatty Acid-Inflammation Relationship

Dietary omega-3 fatty acid (dN3FA) intake affects plasma N3FA (pN3FA) concentrations, circulating biomarkers of chronic inflammation such as high-sensitivity C-reactive protein (hsCRP), and ultimately cardiometabolic disease risk. Genetic variation may modify the N3FA-inflammation relationship, facilitating applications in precision medicine and possibly explaining variable intervention trial responses. Here, we explored the dual hypothesis that pN3FA mediates the dN3FA-hsCRP relationship and that genetic variants across the frequency spectrum modify this association. Regression models in the UK Biobank (N = 342,120) tested main effects of oily fish intake (a primary dietary source of N3FA; measured by food frequency questionnaire) on hsCRP and its mediation by pN3FA (measured by nuclear magnetic resonance; N = 188,701). Three genome-wide interaction studies tested modification of the dN3FA-hsCRP (“full”), dN3FA-pN3FA (“upstream”), and pN3FA-hsCRP (“downstream”) associations. Regression covariates included demographics, lifestyle indicators, socioeconomic measures, and genetic principal components. Variant-specific GWIS were followed by gene-level enrichment testing using the MAGMA tool. Replication of gene-based signals was conducted in the Women’s Genome Health Study (WGHS; N = 23,294) by testing the interaction of the top variant in each gene with total dN3FA impacting hsCRP. Oily fish intake associated most strongly with hsCRP compared to other questionnaire-based estimates of fish or fish oil intake, with statistical evidence that plasma N3FA mediated approximately 100% of this diet-inflammation relationship. While the “full” genetic interaction analysis did not produce any FDR-significant gene-level interactions, the “upstream” analysis uncovered nine genes, including a cluster containing the biologically-relevant fatty acid desaturases FADS1 and FADS2 , and the “downstream” analysis uncovered four genes. Primary variant interaction effect sizes were small (e.g., -0.02 std. dev. bN3FA / std. dev. fish / allele for the top FADS1 variant rs174560), but in-depth follow-up at these genes revealed allelic spectra including rare variants with substantially larger effects (e.g., -0.11 for a FADS1 missense variant with allele frequency of 0.14%, after conditioning on rs174560). WGHS results supported the overall robustness of the findings (consistent interaction effect signs at 8/10 loci), replicated specific signals (p = 0.04 for FADS1 rs174560), and implicated additional inflammatory biomarkers (e.g., fibrinogen and ICAM-1 for the top GLTSCR1 variant). In conclusion, we report (1) the novel epidemiological finding that pN3FA fully mediates the dN3FA-hsCRP relationship, and (2) specific genes with large-effect rare variants that modify the upstream (absorption) and downstream (inflammatory pathways) components of this relationship.

Accurate top protein variant discovery via low-N pick-and-validate machine learning

A strategy to obtain the greatest number of best-performing variants with least amount of experimental effort over the vast combinatorial mutational landscape would have enormous utility in boosting resource producibility for protein engineering. Toward this goal, we present a simple and effective machine learning-based strategy that outperforms other state-of-the-art methods. Our strategy integrates zero-shot prediction and multi-round sampling to direct active learning via experimenting with only a few predicted top variants. We find that four rounds of low-N pick-and-validate sampling of 12 variants for machine learning yielded the best accuracy of up to 92.6% in selecting the true top 1% variants in combinatorial mutant libraries, whereas two rounds of 24 variants can also be used. We demonstrate our strategy in successfully discovering high-performance protein variants from diverse families including the CRISPR-based genome editors, supporting its generalizable application for solving protein engineering tasks. A record of this paper's transparent peer review process is included in the supplemental information.

A top variant identification pipeline for protein engineering

Understanding the fitness of protein variants with combinatorial mutations is critical for effective protein engineering. In this issue of Cell Systems, Chu etal. present TopVIP, a top variant identification pipeline that enables accurate picking of the greatest number of best-performing protein variants with high-fitness leveraging zero-shot predictor and low-N iterative sampling.

Genome-wide association study for the level of prolificacy in Cameroon’s native goat

ABSTRACT Income from goats highly depends on prolificacy, which is difficult to improve by traditional breeding methods. The study aimed to identify SNP markers for prolificacy, using a case–control genome-wide association study (GWAS) on 111 genotyped Cameroon native goat (CNG) does, based on the 50 K single nucleotide polymorphism (SNP) chip panel. None of the top SNPs reached the significant p-value of 5 × 10−8. The highest p-value was 0.0009. Despite the number of cases being about a quarter of the number of controls, the highest allele frequency of some of the top 20 variants in the cases was indicative of their potential role in the trait. These top variants included the following 15: rs268285661, rs268235169, rs268236449, rs268235135, rs268240394 in Sphingosine-1-phosphate phosphatase 2 (SGPP2) gene, rs268283635 in Solute carrier family 24 member 2 (SLC24A2) gene, rs268251678 in Androgen-induced gene 1 (AIG1) gene, rs268267018, rs268239617, rs268281364, rs268273029, rs268286941, rs268236144, rs268233233 in CEP126 gene and rs268278159, respectively. Our findings indicate that GWAS enable the identification of some loci within genes, with known biological functions and pathways in human being and mice animal model but far-ranging to what was previously hypothesized and tested in goat.

Regulatory elements in SEM1-DLX5-DLX6 (7q21.3) locus contribute to genetic control of coronal nonsyndromic craniosynostosis and bone density-related traits

PurposeThe etiopathogenesis of coronal nonsyndromic craniosynostosis (cNCS), a congenital condition defined by premature fusion of 1 or both coronal sutures, remains largely unknown. MethodsWe conducted the largest genome-wide association study of cNCS followed by replication, fine mapping, and functional validation of the most significant region using zebrafish animal model. ResultsGenome-wide association study identified 6 independent genome-wide-significant risk alleles, 4 on chromosome 7q21.3 SEM1-DLX5-DLX6 locus, and their combination conferred over 7-fold increased risk of cNCS. The top variants were replicated in an independent cohort and showed pleiotropic effects on brain and facial morphology and bone mineral density. Fine mapping of 7q21.3 identified a craniofacial transcriptional enhancer (eDlx36) within the linkage region of the top variant (rs4727341; odds ratio [95% confidence interval], 0.48[0.39-0.59]; P = 1.2E−12) that was located in SEM1 intron and enriched in 4 rare risk variants. In zebrafish, the activity of the transfected human eDlx36 enhancer was observed in the frontonasal prominence and calvaria during skull development and was reduced when the 4 rare risk variants were introduced into the sequence. ConclusionOur findings support a polygenic nature of cNCS risk and functional role of craniofacial enhancers in cNCS susceptibility with potential broader implications for bone health.

AAV capsid bioengineering in primary human retina models

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.

Sex‐specific genetic architecture of multiple domains of cognition among older adults

AbstractBackgroundGenetic analyses of cognitive endophenotypes have led to discoveries of novel loci contributing to Alzheimer’s disease (AD) risk. Sex differences are present in cognitive trajectories in aging and AD, and these may vary across cognitive domain. However, genetic drivers that may contribute to sex differences in cognitive trajectories have yet to be explored. Thus, we sought to investigate the sex‐specific genetic architecture of cognition.MethodWe leveraged 10 cohorts of cognitive aging and AD to complete this sex‐aware genetic study (N = 31,800; mean age = 73 yrs.; 55% female). Harmonized cognitive scores for memory, executive functioning, and language were derived using confirmatory factor analysis models. We calculated change in cognitive scores over time using a mixed effects model, to facilitate analysis on cognitive decline. We performed GWAS of baseline score and of estimated rate of decline in each domain and in each cohort separately among non‐Hispanic white (NHW) individuals, adjusting for baseline age and genetic principal components. Then we meta‐analyzed the results.ResultIn addition to the well‐characterized APOE locus, we identified a genome‐wide significant chromosome 2 locus that was associated with language decline among NHW women (rs13387871: MAF = 0.20; βwomen = 2.97×10−3; Pwomen = 2.65×10−9), but not among male counterparts (βmen = ‐3.14×10−4, Pmen = 0.60). This locus contains multiple eQTLs for VRK2, a serine/threonine kinase that has been previously linked to neuropsychiatric disorders, including schizophrenia. Furthermore, the top variant in this locus (rs13387871) was nominally significant for memory decline (βwomen = 1.74×10−3, Pwomen = 0.01) and for executive functioning decline (βwomen = 7.58×10−4, Pwomen = 0.02) in meta‐analyses among NHW women.ConclusionOur genetic analysis suggests that there may be some genetic drivers of language performance that differ by sex, and that these drivers may be shared to an extent across domains. Our future sex‐aware meta‐analyses will also include 1) non‐Hispanic black (NHB) within ancestry (N = 4,200), 2) cross‐ancestry (NHW + NHB), 3) diagnosis‐stratified, and 4) analysis of X‐chromosome. Planned follow‐up analyses will include gene‐set analyses, heritability tests, and genetic correlation tests. Through our preliminary analysis, we identified a promising locus for further exploration, and this is the first of many steps in elucidating the sex‐specific genetic architecture of cognition across ancestry groups.

Amyloid β Induces Lipid Droplet‐Mediated Microglial Dysfunction in Alzheimer’s Disease

AbstractBackgroundSeveral microglia‐expressed genes have emerged as top risk variants for Alzheimer’s disease (AD) that are involved in lipid droplet (LD) formation and processing. Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD‐risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. LD accumulation in aging (Nat Neurosci. 23(2),194‐208, 2020) appears to increase microglial inflammatory responses, while inflammatory challenges increase LD formation, thereby potentially setting up a feed‐forward injury that could contribute to the relentless progression in AD.MethodLD formation is dependent upon age and disease progression, and is more prominent in the hippocampus in brains from human patients and the AD mouse model 5xFAD. Despite variability in LD load between microglia from male versus female animals and between cells from different brain regions, LD‐laden microglia exhibited a deficit in amyloid‐beta (Aβ) phagocytosis evaluated using AβpH ‐ a human Aβ1‐42 analog that exhibits fluorescence upon lysosomal internalization (Chem Sci. 12, 10901‐10918, 2021). Extensive unbiased lipidomic and metabolomic profiling in microglia identified a substantial decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TAGs) as the key metabolic transition underlying LD formation.ResultWe show that microglia form LDs upon exposure to Aβ, and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. Mechanistically, Aβ alone is sufficient to induce LD formation in microglia and that this conversion requires a specific metabolic conversion of FFAs to TAGs (the building blocks of LDs). This conversion is likely protective for the brain and microglia, as we recently showed that long‐chain saturated FFAs can be neurotoxic (Nature 599,102‐107, 2021), but it is not without cost for microglial phagocytic abilities. We demonstrate that a key enzyme for the conversion of FFAs to TAGs, promotes microglial LD formation, is increased in microglia from 5xFAD and human AD brains, and that inhibiting it improved microglial uptake of Aβ.ConclusionThese findings identify a new lipid‐mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.

X‐chromosome wide association study for Alzheimer’s disease

AbstractBackgroundAlthough the X‐chromosome represents about 5% of the genome, it has been excluded from the major genome‐wide association studies on Alzheimer’s Disease (AD) due to its genetic specificities. We performed an in‐depth X‐Chromosome‐Wide Association Study (XWAS) to better characterize the genetic landscape of AD.MethodThe XWAS is based on 49,333 clinically diagnosed cases and 68,882 controls (including 31,314 female cases and 39,407 female controls) of European ancestry from 29 studies from the International Genomics of Alzheimer’s Project (IGAP) and the European Alzheimer &amp; Dementia Biobank (EADB) consortia. 45,993 samples were imputed with the 1000G reference panel and 72,220 samples were imputed with the TOPMed panel.We considered three approaches to take into account the different inactivation scenarios on the X‐chromosome: 1) random X‐chromosome inactivation (r‐XCI) in females, equivalent to an additive genetic model with males coded as homozygous females; 2) skewed X‐chromosome inactivation in females, where a dominance effect was added to the r‐XCI model to capture the inactivation skewedness; 3) escape X‐chromosome inactivation in females, equivalent to an additive model with males coded as hemizygous. For each approach, we performed a single variant analysis with a meta‐analysis of the 29 studies, followed by a gene‐based analysis.ResultWe analyzed 404,688 variants, including 225,045 common variants (Minor Allele Frequency [MAF] &gt; 1%). No genome‐wide significant signals were found in either approach, but three suggestive signals (p‐value &lt; 1×10‐5) were identified in the r‐XCI approach. In one of these loci, we detected an X‐chromosome‐wide significant gene with the gene‐based analysis (p‐value = 5.45×10‐5). The top variant of this locus shows a similar effect in males and females (odds ratio [OR] = 0.93, 95% confidence interval [CI] = 0.90–0.96, p‐value = 8.63 × 10−6, and MAF = 32%, with OR[CI] in females = 0.86[0.79‐0.94], and OR[CI] in males = 0.92 [0.87‐0.97]).ConclusionWe identified promising genetic signals for AD on the X‐chromosome. To increase power, we are currently updating the imputation with TOPMed for all the studies and extending the analyses to large European biobanks.