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Association between copy number variations in the OCA2-HERC2 locus and human eye colour

Published:September 28, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.09.030

      Abstract

      Human eye colour variation is strongly associated with single nucleotide polymorphisms (SNPs) in the OCA2-HERC2 locus, especially rs12913832 that is found in an enhancer element of OCA2. In a previous study we found that 43 out of 166 individuals in a Norwegian population with the brown eye colour genotype HERC2 rs12913832:AA or AG, did not have the expected brown eye colour. To investigate if duplications or deletions in the OCA2-HERC2 locus could explain the blue eye colour in these individuals, we analysed massively parallel sequencing (MPS) data for copy number variations (CNVs) in the OCA2-HERC2 region. The ∼500 kb long OCA2-HERC2 locus was sequenced in 94 individuals with the rs12913832:AG and AA genotypes. Of these, 43 were observed to have blue eye colour and 51 were observed to have brown eye colour. CNVs were analysed using R and the R-package panelcn.MOPS - CNV detection tool for targeted NGS panel data. In rs12913832:AG individuals, CNVs in 32 regions were significantly associated with blue eye colour (Benjamini-Hochberg adjusted p-value ≤ 0.05). In rs12913832:AA individuals, CNVs in 14 regions were associated with blue eye colour using raw p-values (p ≤ 0.05). The functional effects of these CNVs on OCA2 expression are yet to be investigated. However, this study suggests that CNVs in the OCA2-HERC2 locus might explain why some of the rs12913832:AG and AA individuals have unexpectedly blue eyes.

      Keywords

      1. Introduction

      Single nucleotide polymorphisms (SNPs) in the OCA2-HERC2 locus have been extensively studied to explain human eye colour variations. The main predictor for eye colour is the SNP rs12913832 in HERC2 [
      • Sturm R.A.
      • Duffy D.L.
      • Zhao Z.Z.
      • Leite F.P.N.
      • Stark M.S.
      • Hayward N.K.
      • Martin N.G.
      • Montgomery G.W.
      A single SNP in an evolutionary conserved region within intron 86 of the HERC2 gene determines human blue-brown eye color.
      ]. This SNP is located ∼21kb upstream to the promoter of the pigmentation gene OCA2, and acts as a distal OCA2 enhancer [
      • Visser M.
      • Kayser M.
      • Palstra R.-J.
      HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter.
      ]. Individuals with the genotypes rs12913832:AA and AG are predicted to have brown eye colour, whereas individuals with rs12913832:GG are predicted to have blue eye colour [
      • Walsh S.
      • Wollstein A.
      • Liu F.
      • Chakravarthy U.
      • Rahu M.
      • Seland J.H.
      • Soubrane G.
      • Tomazzoli L.
      • Topouzis F.
      • Vingerling J.R.
      • Vioque J.
      • Fletcher A.E.
      • Ballantyne K.N.
      • Kayser M.
      DNA-based eye colour prediction across Europe with the IrisPlex system.
      ]. However, we found that 43/166 individuals in a Norwegian study population with the genotype rs12913832:AA or AG did not have brown eye colour [
      • Salvo N.M.
      • Janssen K.
      • Kirsebom M.K.
      • Meyer O.S.
      • Berg T.
      • Olsen G.H.
      Predicting eye and hair colour in a Norwegian population using Verogen’s ForenSeq™ DNA signature prep kit.
      ].
      The aim for this study was to analyse massively parallel sequencing (MPS) data for copy number variations (CNV) to investigate if duplications or deletions in the OCA2-HERC2 locus could explain the unexpected blue eye colour in rs12913832:AA and AG individuals.

      2. Materials and methods

      The OCA2-HERC2 locus (∼500kb) was sequenced in 94 HERC2 rs12913832:AG and AA individuals with SureSelectXT HS2 Target Enrichment System (Agilent Technologies) on an Illumina MiSeq with paired-end sequencing (2×150bp) using the MiSeq Reagent Kit V2 (300 cycles). Individuals were grouped into a control group with observed brown eye colour (21 AA and 30 AG individuals) and a case group with observed blue eye colour (three AA and 40 AG individuals). All samples were collected with fully informed consent and subsequently anonymised. The project was approved by the Faculty of Health Sciences, UiT - The Arctic University of Norway (reference number 2021/2034).
      All samples were aligned to the GRCh38 human genome assembly using Burrows-Wheeler Aligner, BWA-MEM algorithm [
      • Li H.
      • Durbin R.
      Fast and accurate long-read alignment with Burrows–Wheeler transform.
      ,

      H. Li, Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM, 2013. 〈http://github.com/lh3/bwa〉 (Accessed 31 January 2022).

      ]. SAM files were converted into BAM files using SAMtools [
      • Li H.
      • Durbin R.
      Fast and accurate short read alignment with Burrows–Wheeler transform.
      ]. CNVs were detected in bins of 100 bp with 50 bp overlap, using R and the R-package panelcn.MOPS – CNV detection tool for targeted NGS panel data, with default settings [
      • Povysil G.
      • Tzika A.
      • Vogt J.
      • Haunschmid V.
      • Messiaen L.
      • Zschocke J.
      • Klambauer G.
      • Hochreiter S.
      • Wimmer K.
      panelcn.MOPS: copy‐number detection in targeted NGS panel data for clinical diagnostics.
      ]. The panelcn.MOPS assign all case samples to a CN class per region of interest based on fold changes in read counts (RC) relative to control samples: CN0 (homozygous deletion), CN1 (heterozygous deletion), CN2 (no change), CN3 (heterozygous duplication) and CN4 (homozygous duplication). All control samples were set to have two copies throughout the investigated region (CN2). Association with eye colour was tested using Fisher’s exact test.

      3. Results

      When comparing CNV frequencies between the blue and brown eye colour category, CNVs in 32 regions were statistically significantly associated with blue eye colour in rs12913832:AG individuals (Benjamini-Hochberg adjusted p-value ≤ 0.05). A total of 25 of these regions were considered as candidate regions to explain eye colour variation as they were located in OCA2 or close to the OCA2 enhancer SNP, rs12913832 in HERC2 (Table 1). Seven CNVs were in HERC2, ∼100 kb upstream rs12913832. Notably, some regions in rs12913832:AG individuals, especially in OCA2 intron 21 and 23, were flagged for low quality (possibly low read counts) in a substantial proportion of the samples (Table 1). Thus, results should be interpreted with care.
      Table 1CNVs detected in each CN class for selected regions that were significantly associated (adjusted p-value ≤ 0.05) with eye colour in rs12913832:AG individuals. The estimated CN classes are based on expected fold changes in RC: 0.025 (CN0), 0.57 (CN1), 1 (CN2), 1.46 (CN3) and 2 (CN4). All control samples (brown) were set to CN2.
      Gene Intron
      Intron annotation from GRCh38, chr.15, NM_000275.3 (OCA2) and NP_000266.2 (HERC2), NCBI.
      StartEnd#Homozygous deletions (CN0)#Heterozygous deletions (CN1)#CN2#Duplications (CN3)#Amplifications (CN4)Adjusted p-value value
      Benjamini-Hochberg adjusted p-value.
      LowQual
      Number of test samples flagged for low quality by panelcn.MOPS.
      BlueBrownBlueBrownBlueBrownBlueBrownBlueBrown
      OCA2 23277605202776061900130253020005.0E-0227/40
      277876492778774800130263010005.0E-0223/40
      277876992778779800130253020004.8E-0223/40
      277877992778789810110253030004.9E-0226/40
      277983762779847500180223000004.9E-0312/40
      277984262779852500140243020002.8E-0211/40
      278398622783996100180203020002.1E-0324/40
      278399122784001100180183040009.2E-0433/40
      278399622784006100160233010001.2E-0229/40
      278400122784011100170213020004.4E-0336/40
      278400622784016100150233020001.5E-0230/40
      278401622784026100140263000004.6E-0211/40
      278402122784031100150253000002.9E-0215/40
      278403122784041100150253000002.7E-021/40
      OCA2 21278585532785865200160243000001.9E-0231/40
      278586532785875200150243010002.1E-0239/40
      278587032785880200140253010003.3E-0240/40
      278587532785885200140263000004.5E-0240/40
      OCA2 19278734202787351900140263000004.3E-020/40
      278925562789265500130263010004.8E-028/40
      278926062789270500130263010004.7E-025/40
      OCA2 14279765562797665500110253040004.7E-0221/40
      OCA2 2280425492804267000302330120202.6E-0232/40
      280674382806755900180213010004.6E-037/40
      HERC2 82281256902812578900140263000004.1E-020/40
      a Intron annotation from GRCh38, chr.15, NM_000275.3 (OCA2) and NP_000266.2 (HERC2), NCBI.
      b Benjamini-Hochberg adjusted p-value.
      c Number of test samples flagged for low quality by panelcn.MOPS.
      In rs12913832:AA individuals, CNVs in 14 regions were associated with blue eye colour using raw p-values (p ≤ 0.05). Deletions were observed in two of these regions in OCA2 (intron 23), whereas both deletions and duplications were observed in 12 regions in HERC2, ∼100 kb upstream rs12913832.

      4. Discussion

      With current prediction models, blue and brown eye colours are predicted with high accuracies [
      • Walsh S.
      • Wollstein A.
      • Liu F.
      • Chakravarthy U.
      • Rahu M.
      • Seland J.H.
      • Soubrane G.
      • Tomazzoli L.
      • Topouzis F.
      • Vingerling J.R.
      • Vioque J.
      • Fletcher A.E.
      • Ballantyne K.N.
      • Kayser M.
      DNA-based eye colour prediction across Europe with the IrisPlex system.
      ]. However, blue eye colour has frequently been observed in individuals with the non-blue rs12913832:AG and AA genotypes in Scandinavian populations [
      • Meyer O.S.
      • Salvo N.M.
      • Kjærbye A.
      • Kjersem M.
      • Andersen M.M.
      • Sørensen E.
      • Ullum H.
      • Janssen K.
      • Morling N.
      • Børsting C.
      • Olsen G.-H.
      • Andersen J.D.
      Prediction of eye colour in scandinavians using the eyecolour 11 (EC11) SNP set.
      ]. OCA2 SNPs such as rs1800407, rs75643330 and rs121918166 can potentially explain this lighter eye colour in some, but not all of these individuals [
      • Meyer O.S.
      • Salvo N.M.
      • Kjærbye A.
      • Kjersem M.
      • Andersen M.M.
      • Sørensen E.
      • Ullum H.
      • Janssen K.
      • Morling N.
      • Børsting C.
      • Olsen G.-H.
      • Andersen J.D.
      Prediction of eye colour in scandinavians using the eyecolour 11 (EC11) SNP set.
      ,
      • Andersen J.D.
      • Pietroni C.
      • Johansen P.
      • Andersen M.M.
      • Pereira V.
      • Børsting C.
      • Morling N.
      Importance of nonsynonymous OCA2 variants in human eye color prediction.
      ]. In this study we observed intronic duplications and deletions in the OCA2-HERC2 locus in blue-eyed individuals, including regions in OCA2 intron 2 (upstream the missense mutations rs1800407, rs121918166 and rs75643330), and regions in HERC2 intron 82 (upstream rs12913832). It is well known that CNVs can have significant impact on human traits, but copy-number-based genome-wide association studies (GWASs) from MPS data is scarce. One large-scale association study found CNV to be associated with hair colour at HERC2 in a British biobank [
      • Fitzgerald T.
      • Birney E.
      CNest: A novel copy number association discovery method uncovers 862 new associations from 200,629 whole-exome sequence datasets in the UK Biobank.
      ]. To the best of our knowledge, we suggest for the first time CNVs in these regions to be candidates to explain eye colour variation in rs12913832:AG or AA individuals with unexpected blue eye colour. However, other studies must confirm the results and it is yet to be functionally investigated if these structural variants have regulatory effects on OCA2.

      5. Conclusion

      Preliminary results from MPS data revealed CNVs in the OCA2-HERC2 region in blue-eyed rs12913832:AG and rs12913832:AA individuals. Despite the small sample size, we identified candidate regions with significantly more deletions or duplications in blue-eyed compared to brown-eyed individuals. These could potentially explain the unexpected blue eyes in these individuals. However, the functional effects of these CNVs on OCA2 expression are yet to be investigated.

      Declaration of Competing Interest

      None.

      Acknowledgements

      The authors would like to thank all participants. The project was funded by UiT-The Arctic University of Norway.

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