Keywords
lumbar disk degeneration, back pain, transcriptomics, genome wide association study
lumbar disk degeneration, back pain, transcriptomics, genome wide association study
Back pain (BP) is a common debilitating condition with a lifetime prevalence of 40% and a major socioeconomic impact1. According to the Global Burden of Disease 2016 study, it leads the list of disabling conditions in many parts of the world2. The greatest biological risk for episodes of severe BP is thought to be lumbar disc degeneration (LDD)3. Rather than a discrete disease, LDD is now considered a continuous lifelong aging process that usually starts in the third decade of life but may also be seen in children4. It is highly heritable5, as well as being influenced by smoking6, age7 and body mass index8. The overall burden of lumbar disc degeneration on magnetic resonance imaging is a strong predictor of BP episodes9–11. There is a clear genetic predisposition to both BP and LDD with estimates of heritability in the range of 30%–70%12–15. Also, these traits have been shown to share genetic risk factors16. However, little is known about the genetic architecture of BP and only three associated genetic loci have been identified17,18.
This work was focused on the locus at chromosome 8 (8q24.21), which was identified in our previous BP studies17,18. The same locus has been reported associated with having lumbar disc herniation (LDH) in Icelanders undergoing surgery for sciatica19. Bjornsdottir et al. additionally reported an association of this region to height, but concluded that the region does not influence LDH through height. They noted that the most highly associated SNP, rs6651255, was linked to expression of the nearby gasdermin (GSDMC) gene in GTEx data20. Further, the immune-related function of gasdermins implied that the inflammatory and immunological consequences of disk herniation may be linked to sciatica symptom severity.
However, closely located SNPs associated with different traits do not always reflect pleiotropy21. Furthermore, in the case of eQTL studies, causal hypotheses generated through single-variant look up are highly likely to be false positives21. Consequently, a variety of co-localization methods have been introduced22–25 to distinguish the two common scenarios: pleiotropy (the same functional genetic variant is associated with both traits) and linkage (two different functional genetic variants, each associated with a different trait, that are in linkage disequilibrium).
The aim of this study was to (1) verify whether the same functional variant at 8q24.21 is responsible for BP and LDH and (2) to study the effects of this variation on related phenotypes. As a result, we offer an alternative explanation to the assertion by Bjornsdottir et al.17 regarding the role of immunity and inflammation in generating symptoms of sciatica and BP due to variation in expression at the locus in between genes CCDC26 and GSDMC.
We used the BP GWAS performed in all genetically confirmed white British UK Biobank participants (N= 453,862) as target data for future analysis17. We used the summary level-data of LDH GWAS from the original paper of deCODE group19. They do not provide full GWAS scan, only the statistics for the significant SNPs from the 8q24.21 region. We used information about 40 SNPs from this locus for which information about effect size, effective and reference alleles and p-value was available (Supplementary Table 1,3 of the work19).
We derived summary statistic of 2,419 complex traits provided by the Neale Lab and 34 traits from the Gene ATLAS database. Summary statistics for gene expression levels were obtained from Westra Blood eQTL26 (peripheral blood, http://cnsgenomics.com/software/smr/#eQTLsummarydata, file: westra_eqtl_data_hg19.zip (hg19)) and the GTEx database27 (14 tissues28, https://www.gtexportal.org/home/datasets, file: GTEx_Analysis_V6_all-snp-gene-associations.tar).
Summary-level mendelian randomization and heterogeneity in dependent instruments (SMR-HEIDI)24 approach was used to test for potential pleiotropic effects of the 8q24.21 region on back pain (BP) and LDH and other complex traits including gene expression levels in certain tissues. SMR analysis provides evidence for pleiotropy, but is unable to define whether both traits are affected by the same underlying causal polymorphism. The latter is specified by a HEIDI test that distinguishes pleiotropy from linkage. Zhang F. and colleagues developed the software SMR tool implementing the SMR-HEIDI methods24.
SMR tool is suitable to analyze pleiotropic association using summary-level data from GWAS and eQTL studies. We conducted two groups of analysis: target trait vs eQTL and target trait vs other complex traits. Summary-level data from BP GWAS17 was used as target (--gwas-summary option) in both cases. For eQTL analysis we used Westra Blood eQTL data (converted to suitable BSED format by developers) and GTEx v7, converted using --make-besd option (smr --eqtl-flist Gtex_tissue_1.txt --make-besd --out Gtex_tissue_1, where Gtex_tissue_1.txt file correspond to summary-level data of selected tissue). In order to apply SMR tool for complex traits we selected the region +-250kb around the target SNP from each complex trait GWAS. After that, we add new columns Probe_ID with trait_name to corresponded SNP subset. Finally, we merge these SNP subsets into one file and converted it in BESD format using --make-besd option.
In both cases we set the minor allele frequency threshold as 0.03 and included in the HEIDI test SNPs inside +-250kb window centered around the target SNP; other parameters used default settings. The LD matrix for the HEIDI test was estimated using PLINK 1.9 software and individual data of white UK Biobank participants, the subset of individuals used to calculate BP GWAS (N=10,000).
Nominal P for SMR test was set at 6e-4 (0.05/89, where 89 is the total number of probes for the gene expression analysis) and 2e-5 (0.05/2,453, where 2,453 is the total number of complex traits used in analysis). For HEIDI analysis we used a conservative threshold of P = 0.05 (P < 0.05 corresponds to the rejection of pleiotropy hypothesis).
We had previously performed the largest-to-date GWAS for chronic BP using the UK Biobank and CHARGE consortium cohorts, with total of 119384 cases and 334478 controls17. Cases were defined as those having BP of duration >3-6 months; controls included those without back pain or with back pain of <3-6 months. We found variants at 8q41.21 locus that showed a genome-wide significant association (OR=1.05, p = 4.4 × 10-13, after genomic control correction)17. The location chr8:130,717,716 is tagged by intergenic variant rs10956487 which is in perfect LD with rs6651255 (r2=1 according to 1000 Genomes EUR samples), the lead SNP in the Icelandic study of LDH by Bjornsdottir et al.19. The strongest association signal for BP is situated near GSDMC gene; yet another protein-coding gene, FAM49B, is located within a 500 kbp window. The summary of the previously reported results and our findings for locus 8q24.21 are shown in Figure 1.
The BP-related traits and genes analyzed in our work are blue. The locus 8q24.21 is connected to associated traits (p-value < 5*10-8) by green arrows. Possible relations between traits, as inferred from previous studies, are marked by grey arrows. Pairwise pleiotropic effects tested by SMR-HEIDI are denoted by a red cross if the test failed, and by a blue check if the test passed. A new detected pleiotropic effect on expression of FAM49B and BP based on SMR-HEIDI results is denoted by the blue arrow. LDH, lumbar disk herniation; SRDP, self-reported disk problems; HBMD, heel bone mineral density t-score.
We used the SMR-HEIDI approach24 to test for potential pleiotropic effects of the 8q24.21 region (rs6651255) on BP and other complex traits including gene expression levels in relevant tissues. SMR analysis provides evidence for pleiotropy, but is unable to determine whether both traits are influenced by the same underlying causal polymorphism. The latter question was addressed by the HEIDI test which distinguishes pleiotropy from linkage. For SMR-HEIDI testing we used as the target trait the BP GWAS previously performed in all genetically confirmed white British UK Biobank participants17.
First of all, we checked relationships between BP and LDH. We found strong evidence that co-association of these traits to 8q24.21 is due to the pleiotropic action of the same functional variant (PSMR=2.8 × 10-7 and PHEIDI = 0.87; extended data29). We further applied SMR-HEIDI to test for pleiotropic effects of the region in the collection of 2,453 traits available from the UK biobank study and GeneAtlas. We found that the BP risk allele of rs6651255 was negatively associated with heel bone mineral density t-score and positively associated with self-reported prolapsed or slipped disc (both FDRSMR<0.01, underlying data30 and extended data29) and also strongly associated with standing height (underlying data30 and extended data29) and several measures of fat-free mass (all FDRSMR<0.001). However, HEIDI tests showed strong evidence against pleiotropic effects of the same functional variant in this region for BP and these anthropometric traits (all PHEIDI < 0.001), while associations with heel bone mineral density t-score and self-reported prolapsed or slipped disc were likely the result of pleiotropy, controlled by the same functional variant (all PHEIDI > 0.05). This suggests that genetic variants influencing structural spine features – the vertebral body and intervertebral disc – provide the link to BP symptoms, supporting previous work in twins highlighting shared genetic influence between spine degeneration and BP16,31.
The unanticipated results were obtained in the study of expression data. We have previously17 demonstrated that variants in LD with the lead SNP at 8q24.1 were located in potential regulatory regions in chondrocytes and osteoblasts (Roadmap Epigenomics Consortium32). In the current study, we tested expression of genes in the 8q24.21 region in all available tissues considered potentially relevant to development of pain (underlying data33), including musculoskeletal, brain and nerve tissue (from GTEx,20). Given the suggestion by Bjornsdottir et al. that the locus may act through regulation of immune function19, we also investigated expression in whole blood using the blood eQTL database26 and five immune cell subsets from CEDAR34. Similar to the Bjornsdottir et al. study19, we observed that rs6651255 is co-associated with expression of GSDMC in skeletal muscle (FDRSMR=0.01, PSMR=0.0001, underlying data30), with the BP-associated C allele of rs6651255 being associated with increased expression of the gene. However, HEIDI results show this overlap to be unlikely due to pleiotropic action of the same functional variant (PHEIDI = 3×10-7, underlying data30 and extended data29). Our findings suggest that GSDMC is not the gene through which the functional variant acts to influence BP. The next strongest co-association was with increased expression of FAM49B in brain anterior cingulate cortex (PSMR=0.004, underlying data30 and extended data29) and decreased expression in cytotoxic T (CD8+) cells (PSMR=0.02, underlying data30 and extended data29), although these associations were only nominally significant (p<0.05), yet not statistically significant after multiple testing correction (FDRSMR=0.18 and 0.28, respectively). All corresponding PHEIDI were > 0.05, indicating these co-associations are likely due to pleiotropy. The product of FAM49B was found to be expressed in multiple tissues including bone, lymph node and spleen; according to Proteomics DB35, it has highest expression in lymphocytes, and was implicated as a key negative regulator of actin dynamics and T-cell activation36. The precise mechanism of which gene is involved in the development of BP is difficult to infer due to lack of eQTL data available for relevant tissues, such as intervertebral disc.
We hypothesize that the locus 8q24.21 exhibits its influence on BP phenotypes via disk and bone remodeling, and not through influence on anthropometrics or pain processing. Our findings suggest that FAM49B and not GSDMC is the most likely mechanism through which the functional variant leads to BP.
Zenodo: Table S1. List of relevant studies of gene expression with available eQTL data. http://doi.org/10.5281/zenodo.371500533
This project contains the following underlying data:
List of relevant studies of expression data.csv (List of relevant studies of gene expression with available eQTL data)
Zenodo: Tables S2-S3. SMR-HEIDI results for 8q24.21 locus between back pain and other phenotypes. [Data set]. http://doi.org/10.5281/zenodo.371502730
This project contains the following underlying data:
Figures S1–S7. SMR-HEIDI analysis results for 8q24.21 locus between BP and selected phenotypes. Zenodo: http://doi.org/10.5281/zenodo.371512029
This project contains the following extended data:
Figure S1.png (SMR-HEIDI analysis results for rs6651255 between BP and LDH)
Figure S2.png (SMR-HEIDI analysis results for rs6651255 between BP and GSDMC expression in skeletal muscle (GTEx v6))
Figure S3.png (SMR-HEIDI analysis results for rs6651255 between BP and FAM49B expression in Brain anterior cingulate cortex BA24 (GTEx v6))
Figure S4.png (SMR-HEIDI analysis results for rs6651255 between BP and FAM49B expression in CD8 cell line (CEDAR))
Figure S5.png (SMR-HEIDI analysis results for rs6651255 between BP and heel bone mineral) density (UKBB).
Figure S6.png (SMR-HEIDI analysis results for rs6651255 between BP and disc problem phenotype (UKBB))
Figure S7.png (SMR-HEIDI analysis results for rs6651255 between BP and height (UKBB))
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).
We thank Denis Gorev, Anna Torgasheva and Eugeny Pakhomov for help with summary level data collection, processing, and analyses. We thank Michel Georges for permission to use the CEDAR results for this work, and for discussion related to CEDAR. We also thank Natalia Aulchenko for help with the manuscript preparation.
Views | Downloads | |
---|---|---|
F1000Research | - | - |
PubMed Central
Data from PMC are received and updated monthly.
|
- | - |
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Molecular mechanisms of back pain
Peer review at F1000Research is author-driven. Currently no reviewers are being invited.
Alongside their report, reviewers assign a status to the article:
Invited Reviewers | |
---|---|
1 | |
Version 1 22 May 20 |
read |
Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list:
Sign up for content alerts and receive a weekly or monthly email with all newly published articles
Already registered? Sign in
The email address should be the one you originally registered with F1000.
You registered with F1000 via Google, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Google account password, please click here.
You registered with F1000 via Facebook, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Facebook account password, please click here.
If your email address is registered with us, we will email you instructions to reset your password.
If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance.
Comments on this article Comments (0)