Mutations in disguise - Wiley Online Library

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Bernie Baum, for the inspiration, and Elvis 'the King' Presley for the interpretation. Disclosure of conflict of interest. The authors state that they have no conflict of ...
Journal of Thrombosis and Haemostasis, 9: 1973–1976

DOI: 10.1111/j.1538-7836.2011.04461.x

COMMENTARY

Mutations in disguise S . D U G A and R . A S S E L T A Dipartimento di Biologia e Genetica per le Scienze Mediche, Universita` degli Studi di Milano, Milan, Italy

To cite this article: Duga S, Asselta R. Mutations in disguise. J Thromb Haemost 2011; 9: 1973–6. See also Zucker M, Rosenberg N, Peretz H, Green D, Bauduer F, Zivelin A, Seligsohn U. Point mutations regarded as missense mutations cause splicing defects in the factor XI gene. This issue, pp 1977–84.

The interpretation of the sequence variants found in genetic screenings represents a major challenge in the molecular diagnosis of genetic diseases. While some null mutations (e.g. nonsense substitutions, out-of-frame insertions or deletions) are easy to interpret, the impact of other variants is more difficult to evaluate, and frequently requires an experimental validation. In most cases, the pathogenic role of a point mutation is scored on the basis of: (i) its predicted effect on the coding sequence; (ii) its impact on well-characterized splice consensus sequences; and (iii) its position within the gene promoter. However, pathogenic variations may lurk within the coding portion of a gene without altering its translational output or stay nested deep within introns, therefore going undetected by conventional screening strategies based on Sanger sequencing of the coding region [1]. Since the first original observations on exonic sequences modulating splicing [2,3], a growing amount of data from many laboratories have suggested that genomic variants outside splice sites, both in coding and non-coding sequences, may indeed alter splicing, either acting on exonic and intronic regulatory elements or activating cryptic splice sites [4]. Actually, the splicing process, far from being a simple cutand-paste affair, is based on the recognition of a set of exon identity elements (the so-called Ôsplicing codeÕ), which include, besides the consensus splice sites, diverse intronic and exonic splicing enhancers (ISEs and ESEs) and suppressors (ESSs and ISSs) serving as binding sites for specific trans-acting regulatory factors to the pre-mRNA [5–7]. While mutations affecting canonical splice sites are relatively easy to interpret, variations impacting on the poorly conserved splicing regulatory sequences are difficult to predict by in silico analyzes, still, they can lead to dramatic effects on pre-mRNA splicing [1]. The interesting study performed by Zucker and colleagues [8], published in this issue of the Journal of Thrombosis and Haemostasis, is an important contribution in the field, as it Correspondence: Stefano Duga, Department of Biology and Genetics for Medical Sciences, University of Milan, Via Viotti 3/5, 20133 Milan, Italy. Tel.: +39 2 50315823; fax: +39 2 50315864. E-mail: [email protected]  2011 International Society on Thrombosis and Haemostasis

reports the identification and the molecular characterization of three apparent missense variants leading to splicing defects by altering predicted ESE motifs within the factor (F)XI gene exons 7, 10 and 14. The disrupting effect of the identified mutations on functional ESEs was elegantly demonstrated by obtaining a partial rescue of wild-type splicing through compensatory mutations increasing the predicted score of the mutated ESEs. This manuscript stresses the importance of inspecting the effect of point mutations at the RNA level, which is not routinely performed after genetic screening. The study by Zucker and colleagues represents the first characterization of mutations affecting ESE elements in coagulation factor genes. Indeed, only a few examples of ÔelusiveÕ mutations impacting on splicing regulation have been up-to-now described in coagulation factor genes (see Table 1, and references therein). In view of the frequent unavailability of biological specimens suitable for RNA extraction, either from patients or from their relatives, the effect of genetic variations on splicing should be evaluated by transfection experiments in human cells with suitable minigene constructs [22]. In order to prioritize variations for experimental validation, it would be crucial to have information on all the regulatory elements that affect splicing and their mechanism of action. Unfortunately, for most genes, there is little knowledge even on the pattern of physiological alternative splicing. Concerning coagulation factor genes, detailed data on the pattern of splicing variants have been reported for FXI [23] and FV [24] genes, revealing the existence of a complex pattern of alternative isoforms, which might be more sensitive to genetic variants than constitutively spliced ones. A surrogate strategy can be the in silico prediction of ESE elements [25] and of splice-site strength, as presented in the manuscript by Zucker et al. [8]. However, although in silico methods are constantly improving, there is still a major gap between predictions and the actual functionality of splicing determinants. Besides obvious consequences in the diagnostic field, knowledge on the actual molecular mechanisms underlying coagulation factor deficiencies may have additional impacts on their management and therapy. First, given that splicing defects, in most cases, lead to premature termination codons, discrimination between mutations leading to ÔnullÕ alleles or to

Exon Exon Exon Exon Exon

4 7 10 14 14

c.325G>A c.616C>T c.1060G>A c.1693G>A nt 2045 G>A

nt865G>C

cga>tga

Intron 2

Exon 7

a>g a>g

Intron 1 Intron 1

G5509>A

Exon 16 a>g

1701G>T

Exon 10

Intron 1

IVS8+268A>G

Intron 8

IVS6-320 A>T

Intron 6

Ala91Thr P188S G336R E547K Arg681His

Gly249Arg



– –



Ala1779Thr

Gln509His





Leu172Gln

52% 6% 6% 22% < 1 U/dL

60 U/dL

< 1%

2% < 1%

NA

72%

< 1%

< 1%

< 0.05 mg/dL

10 mg/dL

< 0.1 g/L

68% 6% 6% 20% NA

53 U/dL

NA

NA NA

NA

73%

8%

NA.

0.0001 mg/dL

14.1 mg/dL

NA

Putative deep intronic (400 bp 3¢ to exon 2) Apparent missense (activation of a cryptic donor splice site) Apparent missense (exon 4 skipping) Apparent missense (ESE inactivation) Apparent missense (ESE inactivation) Apparent missense (ESE inactivation) Apparent missense (interfering with splicing)

Deep intronic (activation of a cryptic pseudo-exon) Apparent missense (activation of a cryptic acceptor splice site) Deep intronic (activation of a cryptic pseudo-exon) Deep intronic (activation of a cryptic pseudo-exon) Apparent missense (activation of a cryptic donor splice site) Apparent missense (interfering with intron 16 splicing) Deep intronic (new donor splice 1.3 kb 3¢ of exon 1) Deep intronic (activation of a cryptic pseudo-exon)

[20,21]

[8]

[19]

[18]

[17]

[16]

Unpublished*

[15]

[14]

[13]

[12]

[11]

FGB, fibrinogen beta chain gene; FGG, fibrinogen gamma chain gene; NA, not available. Mutation names and numbering are reported according to those present in the corresponding database/ original paper. Factor activity/antigen levels are presented according to measurement units reported in the database/original paper. In case of ÔapparentÕ missense variations having a possible effect on splicing, the mutation has been mentioned only if characterized at the molecular level. Besides extensive literature mining, mutations were searched in: (i) The ÔMutations Causing Rare Bleeding DisordersÕ database (http://www.isth.org/default/index.cfm/publications/registries-databases/mutations-rare-bleeding-disorders/), (ii) The Fibrinogen database (http://site.geht.org/site/PratiquesProfessionnelles/Base-de-donnees-Fibrinogene/Database-English-Version/Fibrinogen-variants-Database-_79_.html), (ii) The FV database of Dr. Hans Vos ([email protected]), (iv) The ÔHaemophilia A MutationÕ database (http://hadb.org.uk/), (v) The ÔHaemophilia B MutationÕ database (http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html)and (vi) The FXI database (http:// www.factorxi.org). No deep intronic mutations and ÔapparentÕ missense mutations involving splice sites or regulatory splicing elements [e.g. exonic splicing enhancers (ESE)] were found as a cause of prothrombin, FVII, FIX, combined FV and FVIII, and vitamin-K dependent factors deficiencies. *Unpublished data by Western and colleagues (see the ÔHaemophilia A MutationÕ database).

F13A

FXIII

F10

FX

F11

F8

FVIII

FXI

F5

FV

FGG

5157T>A

Exon 4



[9,10]

IVS1+2076g>a

Fibrinogen FGB

Intron 1

References

Deficiency Involved gene Exon/Intron Nucleotide change Amino acid change Coagulation factor activity Coagulation factor antigen Mechanism

Table 1 List of deep intronic mutations and ÔapparentÕ missense mutations involving splice sites or regulatory splicing elements in coagulation factor genes

1974 S. Duga and R. Asselta

 2011 International Society on Thrombosis and Haemostasis

Puzzling mutations 1975

single amino-acid substitutions might in principle be helpful for inhibitor risk prediction [26]. Second, as it seems plausible that changes in the level of expression and/or in the functionality of splicing factors might exist between individuals, these could influence the phenotypic manifestation of a disease, partially explaining the unclear genotype–phenotype relationships that are frequently observed in coagulation deficiencies [27]. Third, the proof of principle that RNA-based approaches to the rescue of coagulation factor biosynthesis by targeting RNA splicing is feasible [28,29] encourages new genetic therapy protocols, which might overcome some of the classic drawbacks faced by conventional gene therapy, for example tissue specificity, proper regulation of expression and size of the transgene. In conclusion, even being aware of the potential presence of Ônon-obviousÕ variations affecting splicing, differentiation between pathogenic mutations and innocuous polymorphisms is still jeopardized by our incomplete knowledge of the molecular mechanisms involved. This problem is increasingly complicated when dealing with the results of next-generation massively parallel sequencing of targeted genomic regions, of whole exomes or of whole genomes, which are quickly becoming the most affordable and effective strategies to find pathogenic variations in the personalized medicine era. Mutations in disguise You fooled me with your frame ESE is what you schemed Genome knows how you lied to me YouÕre not the way you seemed (….) But we got wise! Acknowledgements The authors wish to thank Florence Kaye, Bill Giant and Bernie Baum, for the inspiration, and Elvis Ôthe KingÕ Presley for the interpretation. Disclosure of conflict of interest The authors state that they have no conflict of interest. References 1 Pagani F, Baralle FE. Genomic variants in exons and introns: identifying the splicing spoilers. Nat Rev Genet 2004; 5: 389–96. 2 Reed R, Maniatis T. A role for exon sequences and splice-site proximity in splice-site selection. Cell 1986; 46: 681–90. 3 Mardon HJ, Sebastio G, Baralle FE. A role for exon sequences in alternative splicing of the human fibronectin gene. Nucleic Acids Res 1987; 15: 7725–33. 4 Baralle D, Baralle M. Splicing in action: assessing disease causing sequence changes. J Med Genet 2005; 42: 737–48. 5 Fu X. Towards a splicing code. Cell 2004; 119: 736–8. 6 Wang G, Cooper T. Splicing in disease: disruption of the splicing code and the decoding machinery. Nat Rev Genet 2007; 8: 749–61.

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7 Witten JT, Ule J. Understanding splicing regulation through RNA splicing maps. Trends Genet 2011; 27: 89–97. 8 Zucker M, Rosenberg N, Peretz H, Green D, Bauduer F, Zivelin A, Seligsohn U. Point mutations regarded as missense mutations cause splicing defects in the factor XI gene. J Thromb Haemost 2011; 9: 1977– 84. 9 Dear A, Daly J, Brennan SO, Tuckfield A, George PM. An intronic mutation within FGB (IVS1+2076 a–>g) is associated with afibrinogenemia and recurrent transient ischemic attacks. J Thromb Haemost 2006; 4: 471–2. 10 Davis RL, Homer VM, George PM, Brennan SO. A deep intronic mutation in FGB creates a consensus exonic splicing enhancer motif that results in afibrinogenemia caused by aberrant mRNA splicing, which can be corrected in vitro with antisense oligonucleotide treatment. Hum Mutat 2009; 30: 221–7. 11 Asselta R, Duga S, Spena S, Peyvandi F, Castaman G, Malcovati M, Mannucci PM, Tenchini ML. Missense or splicing mutation? The case of a fibrinogen Bbeta-chain mutation causing severe hypofibrinogenemia Blood 2004; 103: 3051–4. 12 Spena S, Asselta R, Plate´ M, Castaman G, Duga S, Tenchini ML. Pseudo-exon activation caused by a deep-intronic mutation in the fibrinogen gamma-chain gene as a novel mechanism for congenital afibrinogenaemia. Br J Haematol 2007; 139: 128–32. 13 Castoldi E, Duckers C, Radu C, Spiezia L, Rossetto V, Tagariello G, Rosing J, Simioni P. Homozygous F5 deep-intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet-rich plasma. J Thromb Haemost 2011; 9: 959– 68. 14 Schrijver I, Koerper MA, Jones CD, Zehnder JL. Homozygous factor V splice site mutation associated with severe factor V deficiency. Blood 2002; 99: 3063–5. 15 Guasch JF, Lensen RP, Bertina RM. Molecular characterization of a type I quantitative factor V deficiency in a thrombosis patient that is ‘‘pseudo homozygous’’ for activated protein C resistance. Thromb Haemost 1997; 77: 252–7. 16 Bagnall RD, Waseem NH, Green PM, Colvin B, Lee C, Giannelli F. Creation of a novel donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two haemophilia A patients. Br J Haematol 1999; 107: 766–71. 17 Gitschier J, Wood WI, Tuddenham EG, Shuman MA, Goralka TM, Chen EY, Lawn RM. Detection and sequence of mutations in the factor VIII gene of haemophiliacs. Nature 1985; 315: 427– 30. 18 Millar DS, Elliston L, Deex P, Krawczak M, Wacey AI, Reynaud J, Nieuwenhuis HK, Bolton-Maggs P, Mannucci PM, Reverter JC, Cachia P, Pasi KJ, Layton DM, Cooper DN. Molecular analysis of the genotype-phenotype relationship in factor X deficiency. Hum Genet 2000; 106: 249–57. 19 Guella I, Solda` G, Spena S, Asselta R, Ghiotto R, Tenchini ML, Castaman G, Duga S. Molecular characterization of two novel mutations causing factor XI deficiency: a splicing defect and a missense mutation responsible for a CRM+ defect. Thromb Haemost 2008; 99: 523–30. 20 Board P, Coggan M, Miloszewski K. Identification of a point mutation in factor XIII A subunit deficiency. Blood 1992; 80: 937– 41. 21 Anwar R, Stewart AD, Miloszewski KJ, Losowsky MS, Markham AF. Molecular basis of inherited factor XIII deficiency: identification of multiple mutations provides insights into protein function. Br J Haematol 1995; 91: 728–35. 22 Baralle M, Baralle D, De Conti L, Mattocks C, Whittaker J, Knezevich A, French-Constant C, Baralle FE. Identification of a mutation that perturbs NF1 gene splicing using genomic DNA samples and a minigene assay. J Med Genet 2003; 40: 220–2. 23 Asselta R, Rimoldi V, Guella I, Solda` G, De Cristofaro R, Peyvandi F, Duga S. Molecular characterization of in-frame and out-of-frame

1976 S. Duga and R. Asselta alternative splicings in coagulation factor XI pre-mRNA. Blood 2010; 115: 2065–72. 24 DallÕOsso C, Guella I, Duga S, Locatelli N, Paraboschi EM, Spreafico M, Afrasiabi A, Pechlaner C, Peyvandi F, Tenchini ML, Asselta R. Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern. Haematologica 2008; 93: 1505–13. 25 Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR. ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acids Res 2003; 31: 3568–71. 26 ter Avest PC, Fischer K, Mancuso ME, Santagostino E, Yuste VJ, van den Berg HM, van der Bom JG; CANAL Study Group. Risk

stratification for inhibitor development at first treatment for severe hemophilia A: a tool for clinical practice. J Thromb Haemost 2008; 6: 2048–54. 27 Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004; 104: 1243–52. 28 Hammond SM, Wood MJ. Genetic therapies for RNA mis-splicing diseases. Trends Genet 2011; 27: 196–205. 29 Pinotti M, Bernardi F, Pagani F. RNA-based therapeutic approaches for coagulation factor deficiencies. J Thromb Haemost 2011; doi:10. 1111/j.1538-7836.2011.04481.x.

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