den Ouden, Amber

Derks, Ronny

Haer-Wigman, Lonneke

Lugtenberg, Dorien

Neveling, Kornelia

van Genderen, Maria M.

Verheij, Joke

Vijzelaar, Raymon

Vissers, Lisenka E.L.M.

Yntema, Helger G.

Utrecht University Repository

npj | genomic medicine Matters arisingPublished in partnership with CEGMR, King Abdulaziz Universityhttps://doi.org/10.1038/s41525-024-00409-9Reply to: Pitfalls in the genetic testing ofthe OPN1LW-OPN1MW gene cluster inhuman subjectsCheck for updatesLonneke Haer-Wigman1,2 , Amber den Ouden1, Ronny Derks1, Maria M. van Genderen 3,4,Dorien Lugtenberg1,2, Joke Verheij5, Raymon Vijzelaar6, Helger G. Yntema1,2, Lisenka E. L. M. Vissers1,2 &Kornelia Neveling1,2REPLYING TO Wissinger, B. npj Genomic Medicine https://doi.org/10.1038/s41525-024-00406-y (2024)We recently published a genetic assay for theOPN1LW/OPN1MW genecluster, comprising four amplicon-based long-read sequencing reac-tions and one MLPA1. Wissinger et al. brought to our attention that, forone of the four amplicons, the primer overlaps with a polymorphism,designated SDIns2. Consequently, the amplicon would be non-specific,from which they claim that our approach will render misleading results.We performed several analyses to disprove this claim. Firstly, it couldonly lead to wrong results if this amplicon requires assessment. Inpractice, retrospectively evaluated from 155 male probands, thisamplicon requires analysis in only 8% of probands. Moreover, errors inthe genetic composition would require a certain distribution of SDInsacross the OPN1LW/OPN1MW cluster, which, based on the analysis of200 alleles using optical genome mapping, does not seem to occur in thepopulation. Indeed, long read genome sequencing, in combination withde novo assembly for two cases from the original publication, confirmedour original findings. Lastly, it is not always necessary to know thecomplete composition of the cluster to conclude whether or not theclinical diagnosis is genetically confirmed. In all 8% probands in whomthis amplicon would be analyzed an incorrect determination of thecomposition of the cluster would have resulted in the same conclusion ofthe genetic assay. Taken these results together, the genetic assay for theOPN1LW/OPN1MW gene cluster as described in our previous paper1allows for a clinically relevant determination of the composition of theOPN1LW/OPN1MW gene cluster.With interest, we took notice of the manuscript by Wissinger andcolleagues2. Indeed, as highlighted by the authors, the reverse primerto amplify the last opsin gene copy is located within the segmentalduplicated region of OPN1MW. This segmental duplication region is,however, interrupted by non-repeat sequence (Fig. 1): a 697 base pairinsertion polymorphism (denoted as SDIns by Wissinger et al.).Although this SDIns is exclusively reported to be downstream of thesecond OPN1MW gene copy in all three available reference genomesGRCh37/hg19, GRCh38 and T2T-CHM13, Ueyama et al. alreadypublished in 2004 its occurrence and distribution across the opsingene cluster in detail and highlighted the benefits of using the SDInsfor characterization of the OPN1LW/OPN1MW cluster3. Wissingeret al. confirms that this SDIns is not exclusively located downstream ofthe last opsin gene copy2, but challenges its use to characterize theOPN1LW/OPN1MW cluster because of non-specific amplification,and thus also challenges our approach.In our approach1, the amplicon for the last opsin gene copy onlyrequires sequencing in patients with three opsin gene copies in whomthe last two opsin gene copies are not identical, as in patients withthree or more opsin gene copies in which the second and consecutivegene copies are identical, the exact order is redundant. Also, as alreadystated our approach cannot determine the composition of the com-plete cluster when the cluster contains four or more opsin gene copiesand the second and consecutive gene copies are not identical1. Weaimed to assess the impact ofWissinger et al.’s notions by determiningthe number of patients in his cohort fulfilling the above criteria, butcannot do so as essential information on the complete make-up of theOPN1LW/OPN1MW gene cluster of the samples in their cohort anddetailed methods are lacking2. We therefore revisited the genetic testresults of a cohort of 155 male probands for whom genetic testing ofthe OPN1LW/OPN1MW gene cluster was performed, including 33 ofour original report1. Of these 155 patients, 70 (45%) had three or moreopsin gene copies in their cluster, but only 13 (8%) had three opsincopies where the last two copies differed, putting them at risk forerrors in the genetic outcome of our approach (Supplementary Table 1,Supplementary Fig. 1).To comprehensively assess this risk, it is, however—in addition to theabsolute copy number of SDIns—also essential to understand the distribu-tion of SDIns over the different opsin gene copies. WhereasWissinger et al.determined the copy number of the SDIns using qPCR, positional1Department of HumanGenetics, RadboudUniversityMedical Center, Nijmegen, TheNetherlands. 2Research Institute forMedical Innovation, RadboudUniversityMedical Center, Nijmegen, The Netherlands. 3Bartiméus Diagnostic Center for complex visual disorders, Zeist, the Netherlands. 4Department of Ophthalmology,University Medical Centre Utrecht, Utrecht, the Netherlands. 5Department of Genetics, University Medical Center Groningen, University of Groningen,Groningen, the Netherlands. 6MRC Holland b v, Amsterdam, the Netherlands. e-mail: lonneke.haer-wigman@radboudumc.nlnpj Genomic Medicine |            (2024) 9:29 11234567890():,;1234567890():,;information cannot be determined from their approach2, as this wouldrequire single longmolecules spanning the distance of the entireOPN1LW/OPN1MW cluster. We, therefore, used optical genome mapping4 to deter-mine the location of SDIns in 200 alleles from random individuals (colorvision status unknown) with three opsin gene copies (Supplemental Fig. 3).This analysis showed that in the vast majority of alleles (153 of 200; 76.5%),SDIns was located solely after the last opsin gene copy (Fig. 2a); in 12 alleles(6%), SDInswas present after thefirst and last opsin gene copy (Fig. 2b). Forboth situations, collectively representing 82.5% of individuals with threecopies of the opsin cluster, a correct diagnosis would be obtained using ouroriginally reported approach. In 35 alleles (17.5%) the amplicon wouldrender an inconclusive genetic result due to overlapping sequences or noPCR products (Fig. 2c–f). Most importantly, the two possible options thatwould lead to an incorrect genetic result, e.g. the presence of the SDIns afterthe second opsin gene copy and the absence of the SDIns after the thirdopsin gene copy, were absent from the 200 investigated alleles (Fig. 2g, h).Therefore, extrapolating these results from the population suggests that thelikelihood of an incorrect determination of the composition of the cluster isnegligible. However, as the majority of alleles analyzed is probably fromindividuals of a Caucasian background, there is a possibility that the dis-tribution of the SDIns differs in populations with other ethnicities. Of note,as an additional verification, we performed long-read genome sequencingfor two of the clinical cases from the original study1, which for bothconfirmed the genetic compositionof theOPN1LW/OPN1MW gene clusteras reported (Supplemental Fig. 2).Despite the unlikely scenario of an incorrect determination ofOPN1LW/OPN1MW gene cluster, we assessed whether or not this wouldimpact the clinical diagnosis. In all 13 probandswith three opsin gene copiesof which the last two were different, sequencing of the amplicon for the firstopsin gene copy, and the amplicon for the second and if present consecutiveopsin gene copies were already enough for a conclusive test result, irre-spective of it being positive, inconclusive or negative. Thus, the result for theamplicon for the last gene copywould refine the composition, butwouldnotchange the conclusion.The discussion on strategies to evaluate the OPN1LW/OPN1MW genecluster highlights the complexity of the locus. Whereas we agree with Wis-singer et al. that, ideally, (ultra-)long read (genome) sequencingwith de novoassembly would help to resolve these challenges, such strategy is not yetavailable in routine diagnostic settings, due to the relative high costs (longread circular consensus sequencing developed by Pacific Biosciences), orrelativehighperbase error rate (long readnanopore sequencingdevelopedbyOxford Nanopore)5. Having shown that SDIns has no impact our on diag-nostic outcomes, and that analysis of the third copy ofOPN1LW/OPN1MWis obsolete for clinical interpretation, we therefore remain of the opinion thatat themoment, the genetic test described byHaer-Wigman et al.1 is themostcomplete diagnostic test for the OPN1LW/OPN1MW gene cluster.Fig. 1 | Print screens of UCSC genome track of the OPN1LW/OPN1MW genecluster in different genome assemblies. Three different genome assemblies areshown, GRCh37/hg19 in (a), GRCh38 in (b), and T2T in (c). In all three panels, thelocation of the reverse primers sequence (099-821, TCTCATTCA-TAAATTGCTGGTA) of the amplicon for the last opsin gene in the cluster isdepicted by the light blue bar. Although the primer sequence is located within thesegmental duplicated region in theGRCh37/hg19,GRCh38, andT2T assemblies, theprimer sequence is uniquely aligned in all three genome builds. The human chainedself-alignment track (only available for GRch37/hg19 and GRCh38 assemblies)visualizes the fact that the segmental duplication is interrupted and the primer islocated in the non-repeat region. In GRCh37 and T2T, the OPN1LW/OPN1MWgene cluster consists of three opsin gene copies and primer 099-821 is located behindthe last opsin gene copy of the cluster (a, c), while in GRCh38, the OPN1LW/OPN1MW gene cluster consists of four opsin gene copies and primer 099-821 islocated behind the second-last opsin gene copy of the cluster (b).https://doi.org/10.1038/s41525-024-00409-9 Matters arisingnpj Genomic Medicine |            (2024) 9:29 2Reporting summaryFurther information on research design is available in the Nature ResearchReporting Summary linked to this article.Data availabilityThe individual-level sequencing and optical genome mapping data areavailable behind the Radboudumc firewall. In the absence of explicit data-sharing consent at the individual patient level, FASTQ, BAM, and VCFscannot be disclosed. These data are, however, available for review at or via asecured connection with the Department of Human Genetics of the Rad-boudumc. Data is available upon reasonable request and after a data usageagreement through the corresponding author L.H-W.Received: 26 September 2023; Accepted: 13 March 2024;References1. Haer-Wigman, L. et al. Diagnostic analysis of the highly complexOPN1LW/OPN1MW gene cluster using long-read sequencing andMLPA. NPJ Genom. Med. 7, 65 (2022).2. Wissinger BB, B., Buena-Atienza, E., Gross, C. & Kohl, S. Pitfalls in thegenetic testing of the OPN1LW-OPN1MW gene cluster in humansubjects. NPJ Genom. Med. https://doi.org/10.1038/s41525-024-00406-y (2024).3. Ueyama, H., Torii, R., Tanabe, S., Oda, S. & Yamade, S. An insertion/deletion TEX28 polymorphism and its application to analysis of red/green visual pigment gene arrays. J. Hum. Genet. 49, 548–557 (2004).4. Chan, S. et al. Structural variation detection and analysis usingbionano optical mapping. Methods Mol. Biol. 1833, 193–203(2018).5. Logsdon, G. A., Vollger, M. R. & Eichler, E. E. Long-read humangenome sequencing and its appliations. Nat. Rev. Genet. 21,597–614 (2020).Author contributionsConceptualization: L.H.-W. and K.N.; Investigation: A.O., R.D.;Methodology: A.O., R.D, Supervision: H.G.Y., L.E.L.M.V; Visualization:L.H.-W. and K.N.; Writing-original draft: L.H.-W, K.N.; Writing-review &editing: M.M.G., D.L., J.V., R.V., H.G.Y., L.E.L.M.V. All authors edited andhave read and approved the manuscript.Competing interestsThe authors declare no competing interests.Additional informationSupplementary information The online version containssupplementary material available athttps://doi.org/10.1038/s41525-024-00409-9.Correspondence and requests for materials should be addressed toLonneke Haer-Wigman.Reprints and permissions information is available athttp://www.nature.com/reprintsabcdefghCorrectCorrectInconclusiveInconclusiveInconclusiveInconclusiveIncorrectIncorrectDifferent possibilities of SDIns location Genetic ResultFrequency76.5%6%10%1.5%5%1%0%0%Fig. 2 | Schematic representation of all possible locations of the SDIns (bluetriangle) in a three opsin gene copy (dark grey bars) cluster. The figure shows thefrequency of the specific composition determined in 200 alleles of individuals (bothmale and female) whose color vision status was unknown, plus whether the specificcomposition would lead to a correct inconclusive or incorrect result. a In 76.5% (135alleles) the SDIns are solely located behind the last opsin gen copy and the geneticassay would correctly determine the composition of the cluster. b In 6% (12 alleles)the polymorphismcan be found downstreamof thefirst and of the last gene. Also, forthis situation, the genetic assay would correctly determine the composition of thegenetic cluster, as with long-read sequencing one can easily differentiate between thefirst and last gene copy using the result of the amplicon specific for thefirst gene copy.In 17.5% (35 alleles) the genetic assays would not be able to determine the exactcomposition of the cluster as the amplicon for the last opsin gene copy wouldsequence both the second-to-last and last opsin gene copy c, d or neither the second-to-last and last opsin gene copywould be amplified. e, fThese last four options, wouldnot lead to an incorrect genetic diagnosis as the sequencing results clearly show aninconclusive result: either no or two alleles are sequenced. g, h The two possibilitieswhere an incorrect genetic result would be determined with the genetic assay. Thissituation could, however, not be detected in 200 alleles with three opsin gene copies.https://doi.org/10.1038/s41525-024-00409-9 Matters arisingnpj Genomic Medicine |            (2024) 9:29 3Publisher’s note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in anymedium or format, as longas you give appropriate credit to the original author(s) and the source,provide a link to the Creative Commons licence, and indicate if changeswere made. The images or other third party material in this article areincluded in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in thearticle’sCreativeCommons licence and your intended use is not permittedby statutory regulation or exceeds the permitted use, you will need toobtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2024https://doi.org/10.1038/s41525-024-00409-9 Matters arisingnpj Genomic Medicine |            (2024) 9:29 4

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