Splicing mutations are less commonly reported than other mutation types1-3. However, despite most being functional nulls, previous reports suggested that they are under-recognized2. Our recent experience of investigating a cohort of 38 individuals with a severe, genetically heterogeneous Mendelian phenotype shows that this continues to be a problem; three mutations that affected splicing were initially “missed” because they were not detected by current splice site detection algorithms. Our concern is that splicing mutations will continue to be overlooked in clinical laboratory settings because the quantity of data generated per person by “exomes” and “genomes” necessitates the use of splice site detection programs. Our cases highlight significant deficiencies in current standard programs, where variants at the U2 canonical AG (acceptor) and GT (donor) splice sites are reliably detected, but variants at other positions with more loosely defined consensus sequences, or U12 splice sites, are rarely detected4.MRC CASE and Wellcome Trust Coll.awar

Nahorski, michael

raj, harjeet

Shaikh, samiha

Woods, CG

Apollo (Cambridge)

European Journal of Human Geneticshttps://doi.org/10.1038/s41431-018-0214-3VIEWPOINTBefore progressing from “exomes” to “genomes”… don’t forgetsplicing variantsSamiha S. Shaikh1 ● Michael S. Nahorski1 ● Harjeet Rai2 ● C. Geoffrey Woods 1Received: 16 October 2017 / Revised: 16 May 2018 / Accepted: 22 May 2018© The Author(s) 2018. This article is published with open accessSplicing variants are less commonly reported than othervariant types [1–3]. However, despite most being func-tional nulls, previous reports suggested that they areunder-recognized [2]. Our recent experience of investi-gating a cohort of 38 individuals with a severe, geneticallyheterogeneous Mendelian phenotype shows that thiscontinues to be a problem; three variants that affectedsplicing were initially “missed” because they were notdetected by current splice site detection algorithms. Ourconcern is that splicing variants will continue to beoverlooked in clinical laboratory settings because thequantity of data generated per person by “exomes” and“genomes” necessitates the use of splice site detectionprograms. Our cases highlight signiﬁcant deﬁciencies incurrent standard programs, where variants at the U2canonical AG (acceptor) and GT (donor) splice sites arereliably detected, but variants at other positions with moreloosely deﬁned consensus sequences, or U12 splice sites,are rarely detected [4].We analyzed 38 sequentially ascertained samplesfrom individuals who were born unable to feel painwithin an UK NHS genetic service. We initially found28 of the 38 cases had bi-allelic variants that affectedfunction in SCN9A (17), NTRK1 (13) and NGF (1);all causing autosomal recessive painless disorders.Given the speciﬁc phenotype and limited genotypes, wehand-curated the remaining cases. In three unrelatedindex individuals rare variants within intronic regionswere present on sequencing (ExAC frequencies of2/113990, 0/61864, and 2/120742). Splice site predictionprogram analysis of each variant was performed usingAlamut (http://www.interactive-biosoftware.com/alamut-visual/), which incorporates ﬁve different programs allof which have been developed for U2 splice sites rather thanU12 splice sites (see Table 1). Whilst the three variantswere not ﬂagged, we considered they could alter splice sitefunction due to their proximity to known splice sites, andassessed each by a minigene splicing assay [5], as tran-scriptomic sequencing was not possible (see Supplement formethodology).Each variant was proven to alter splicing by comparingthe results to those of normal wild-type splicing (see Fig. 1).The ﬁrst case had a hereditary sensory andautonomic neuropathy type 4 (HSAN4) phenotype butsequencing analysis had detected no variants, howeverwe noted a homozygous NTRK1 variant, c.575–19 G >A(reference sequence NM_002529.3; exons are numberedas in the reference sequence NG_007493.1) [6]. Bioinfor-matics analysis suggested that this potentially created anew AG splice acceptor site, which was predicted to besuperior to the existing site; this was the case, withthe introduction of a 17 bp frame-shifting insertion intothe NTRK1 mRNA (Fig. 1b.ii). The second case alsohad a HSAN4 phenotype but sequencing had revealedonly one NTRK1 heterozygous variant proven to affectfunction c.1550 G > A6. On sequence inspection we noted aheterozygous splice donor site variant c.717+ 4 A > T.Although usually+ 4 can be any base, in NTRK1 this+ 4position is invariant [7]. The minigene assay showedthat the variant resulted in complete loss of NTRK1 exon 6in the mature transcript (Fig. 1b.iii). The third case had aphenotype consistent with congenital insensitivity to pain(CIP)—a lack of pain and smell perception withnormal intelligence. Only a single heterozygous variant inSCN9A was detected, c.2686 C > T (reference sequence* C. Geoffrey Woodscw347@cam.ac.uk1 Department of Medical Genetics, Cambridge Institute for MedicalResearch, Hills Road, Cambridge CB2 0XY, United Kingdom2 East Anglian Medical Genetics Service, Genetics Laboratories,Addenbrooke’s Treatment Centre, Cambridge UniversityHospitals, Hills Road, Cambridge CB2 0QQ, United KingdomElectronic supplementary material The online version of this article(https://doi.org/10.1038/s41431-018-0214-3) contains supplementarymaterial, which is available to authorized users.1234567890();,:1234567890();,:NM_002977.3; exons are numbered as in the referencesequence NG_012798.1). On inspection of the sequencingdata we noted a heterozygous variant c.377+ 5 C > Tin SCN9A occurring in a U12 splice site. The U12 donorsite sequence is RTATCCTT where +5 C is invariant [8],in contrast to the more ubiquitous U2 splice site where +5can vary [7]. The variant caused complete loss of exon 3and aberrant splicing into a cryptic U2 acceptor siteresulting in a +1 frame shift (Fig. 1c.ii). All three variantswere predicted to lead to nonsense-mediated decayand hence be nulls. Early nonsense and frame shift variantshave been identiﬁed in other cases of HSAN4 andCIP patients and hence are likely to explain the diseasein our patients [9].In our cohort, a tenth of cases harbored missed variantsthat affected splicing. Their detection was considerablyaided by the clear phenotype and the limited number ofgenes that required analysis. Had the phenotype been morevariable, or did not resemble a single-gene disorder, or thenumber of potentially causative genes greater, it is possiblethat these variants would have gone undetected. These casesalso illustrate the need to consider seeking a second variantin autosomal recessive phenotypes when only a hetero-zygous variant is found; generally the chance of there beinga second variant is greater than the chance the person is anincidental carrier.As the volume of genetic data generated per personcontinues to increase (from exon-by-exon analysis,through gene panels, to exomes, and now whole genomesequencing), this has inevitably led to a greater relianceon variant detection programs. The limitations of thesealgorithms to detect splicing variants, especially thoseoccurring in U12 introns and less well deﬁned consensussequences, needs to be better recognized and urgentlyremedied (for instance, by the use of the Spliceman pro-gram, see Table. 1), otherwise, the full potential of genetictesting will be limited [10]. Until then, researchers inclinical laboratories should be vigilant in seeking splicingvariants and perhaps should hand-curate for rare varia-tions occurring beyond −1, −2, +1, +2 sites. If splicingvariants that affect function are missed by splicing pre-diction programs, or by a conservatism to prevent theidentiﬁcation of too many variants of unclear signiﬁcancein clinical laboratories, then this has two important con-sequences. Firstly, it decreases the utility of exome/gen-ome scale sequencing, and secondly, it increases the riskthat other variations may be erroneously regarded asdisease-causing.Table 1 Summary of the resultsof splice prediction programs fordetecting the wild-type splicesites, and the effects of variantson eachWas the variant predicted to alterthe splice site byNTRK1 c.575–19 G > AExon 6 acceptor siteNTRK1 c.717+ 4 A> T Exon 6 donorsiteSCN9A c.377+ 5 C> T Exon 3 donorsiteMaxEntScan (1–16) Yes: 5 to 3.7 Yes: 7.2 to 1.7 No: NDSpliceSiteFinder-like (0–100) No: 75 to 75 No: 81 to 71 No: NDHuman Splicing Finder (0–100) No: 82.5 to 82.5 No: 91 to 82 No: NDGeneSplicer (0–15) No: 2.5 to 2.5 Yes: 9.2 to 2.5 No: NDNNSplice (0–1) No: 0.9 to 0.9 Yes: 0.7 to 0 No: NDSpliceman (0–100) Yes: 75 Yes: 55 Yes: 82The Alamut program, which incorporates MaxEntScan, SpliceSiteFinder-like, Human Splicing Finder,GeneSplicer and NNSplice, was used as the primary assessment tool for the intronic variants. For eachprediction program in Alamut we have indicated the scale range in brackets and have stated whether thevariants were predicted to be deleterious in bold if the change in variation was greater than 15% (yes/no) andgiven the score of the wild-type splice site and the effect of the variant below the prediction. At least three ofthe ﬁve programs had to strongly predict an effect on splicing at the canonical splice site in order to beconsidered as disease-causing by the clinical laboratory.The NTRK1 c.575–19 G > A variant was only predicted to have deleterious effects on the wildtype acceptorsite by one program, MaxEntScan. The NTRK1 c.717+ 4 A > T variant had deleterious predictions in threeprograms but as the +4 position was known to be the least conserved it was not ﬂagged as being likely toaffect function. The SCN9A exon 3 splice site was not detected as a splice site by any of the ﬁve programs inAlamut, and hence the effect of the variant could not be determined. This was unsurprising as the intron 3 ofSCN9A is a U12 intron, for which none of the Alamut programs were designed. However, the more recentlywritten splice prediction program Spliceman did predicted the splice site and the deleterious effect of thevariant. Spliceman reports variants as a percentage; the higher the percentile rank, the more likely it is thevariation will disrupt splicing.ND: wildtype splice was not detected by the programS. S. Shaikh et al.43 44 45 46PvuI PvuINTRK1 Exon 6SCN9A Exon 3 SCN9A Exon 4USR13-v1AB.iB.iiiC.iiB.iiC.iFig. 1 Summary of minigene assay and sequencing results demonstrating the functional consequences of three “missed” splicing variants. TheUSR13-v1 vector, used for minigene assays, (a) contains COL2A1 exons 43–46 and intervening introns. Between exon 44 and 45, a multiplecloning site is present to allow cloning of the test region. NTRK1 exon 6 and ﬂanking intronic regions were introduced into the vector. SCN9A exon3, intron 3–4 and exon 4, as well as ﬂanking intronic regions were introduced into the vector. Exons are shown as colored boxes; blue and green forthe minigene exons (exon 44 and 45 of COL2A1), orange for NTRK1 exon 6, and grey and purple for SCN9A exons 3 and 4. Mini-gene constructsof wild-type and mutant NTRK1 exon 6 (b) and SCN9A exon 3 and 4 (c), with their ﬂanking intronic regions, were expressed in HeLa cells. PCRwas performed on cDNA converted from extracted mRNA and sequenced. For each reaction the left panel is a schematic of the splicing event andthe right panel an annotated chromatogram of sequence from the minigene PCR reaction. The splice acceptor and donor site nucleotides are shownsurrounding the exons studied, with the invariant nucleotides enlarged. The variants investigated are shown in red. Black arrows indicate normalsplicing. For each variant, a loss of a black arrow indicated that splicing at that site failed to occur, and red arrows indicate new splice sites formedbecause of the variants. b.i details the normal splicing of NTRK1 exon 6. b.ii shows the effect of c.575–19 G >A producing a novel splice acceptorsite 19 bp from the start of exon 6 - resulting in the addition of 17 nucleotides into the mRNA, a frameshift and a premature stop codon. b.iii showsthe effect of c.717+ 4 A > T, a splice donor variant at +4 bp: the whole of exon 6 is missing from the ﬁnal transcript resulting in a frame-shift inthe reading frame of the mRNA and a premature stop codon. c.i details the normal splicing event for SCN9A exon 3 and 4, where intron 3 hasU12 splicing. c.ii shows the effect of c.377+ 5 C > T in the +5 site in the U12 donor site sequence of SCN9A intron 3. This lead to a completefailure of splicing of exon 3, and the use of a cryptic U2 splice donor site prior to the U12 splice acceptor site of exon 4 resulting in 4 bp of intron 3being added to exon 4. This resulted in a+ 1 frame shift, a stop codon at the 21st codon of exon 4, and nonsense-mediated decayBefore progressing from “exomes” to “genomes”… don’t forget splicing variantsAcknowledgements SS acknowledges the support of a MRC CASEstudentship MR/K017551/1, and MSN the support of a WellcomeTrust Collaborative award 200183/Z/15/Z.Compliance with ethical standardsConﬂict of interest The authors declare that they have no conﬂict ofinterest.Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in any medium or format, aslong as you give appropriate credit to the original author(s) and thesource, provide a link to the Creative Commons license, and indicate ifchanges were made. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is notincluded in the article’s Creative Commons license and your intendeduse is not permitted by statutory regulation or exceeds the permitteduse, you will need to obtain permission directly from the copyrightholder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.References1. Yang Y, Muzny DM, Reid JG, et al. Clinical whole-exomesequencing for the diagnosis of mendelian disorders. N EnglJ Med. 2013;369:1502–11.2. Baralle D, Baralle M. Splicing in action: assessing disease causingsequence changes. J Med Genet. 2005;42:737–48.3. Lynch M. Rate, molecular spectrum, and consequences of humanmutation. Proc Natl Acad Sci Usa. 2010;107:961–8.4. Grozeva D, Carss K, Spasic-Boskovic O, et al. Targetednext-generation sequencing analysis of 1000 individualswith intellectual disability. Human Mutat. 2015;36:1197–204.5. Spickett C, Hysi P, Hammond CJ, et al. Deep intronic sequencevariants in COL2A1 affect the alternative splicing efﬁciency ofexon 2, and may confer a risk for rhegmatogenous retinaldetachment. Hum Mutat. 2016;37:1085–96.6. Shaikh SS, Chen YC, Halsall SA, et al. A Comprehensive func-tional analysis of NTRK1 missense mutations causing hereditarysensory and autonomic neuropathy type IV (HSAN IV). HumMutat. 2017;38:55–63.7. Zhang MQ. Statistical features of human exons and their ﬂankingregions. Hum Mol Genet. 1998;7:919–32.8. Clark F, Thanaraj TA. Categorization and characterization oftranscript-conﬁrmed constitutively and alternatively splicedintrons and exons from human. Hum Mol Genet. 2002;11:451–64.9. Indo Y. Molecular basis of congenital insensitivity to pain withanhidrosis (CIPA): mutations and polymorphisms in TRKA(NTRK1) gene encoding the receptor tyrosine kinase for nervegrowth factor. Hum Mutat. 2001;18:462–71.10. Lewandowska MA. The missing puzzle piece: splicing mutations.Int J Clin Exp Pathol. 2013;6:2675–82.S. S. 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Viewpoint: Before progressing from “exomes" to “genomes”… don't forget splicing mutations

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Viewpoint: Before progressing from “exomes" to “genomes”… don't forget splicing mutations

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