splice site

10
Regular Article THROMBOSIS AND HEMOSTASIS Intron retention resulting from a silent mutation in the VWF gene that structurally inuences the 59 splice site Hamideh Yadegari,* Arijit Biswas,* Mohammad Suhail Akhter, Julia Driesen, Vytautas Ivaskevicius, Natascha Marquardt, and Johannes Oldenburg Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany Key Points This study demonstrates allosteric RNA structure alteration resulting from an exonic variation, thereby interfering with splicing. This study details a novel mechanism by which silent mutation distant to the 59 splice site could still result in intron retention. Disease-associated silent mutations are considered to affect the accurate pre– messenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 59 splice site (59ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 59ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 59ss. This hairpin sequesters the 59ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA. (Blood. 2016;128(17):2144-2152) Introduction von Willebrand factor (VWF) is a multimeric plasma glycoprotein synthesized in endothelial cells and platelet precursors and plays crucial roles in hemostasis. 1 The VWF gene (VWF) is composed of 52 exons spanning ;178 kb of the genome, and it is transcribed into an 8.8-kb messenger RNA (mRNA). 2 Decient VWF results in von Willebrand disease (VWD), which is classied as quantitative (type 1 and type 3) or qualitative (type 2). 3,4 Type 1 VWD, characterized by partial reduction in VWF levels, is the most common form of the disorder. 5 Inheritance of type 1 VWD is considered to be autosomal dominant; however, in ;15% of index cases, more than a single candidate VWF variant is detected. 6-8 Pre-mRNA splicing is regulated by consensus core splicing sites comprising the 59 splice site (59ss), the 39 splice site (39ss), and the branch point sequences. 9 The splicing reaction is initiated by RNA- RNA base pairing of the consensus 59ss motif and the U1 small nuclear RNA (snRNA) terminus, which is followed by excision of the 59 end of the intron, ligation of the adjacent exons, and releasing of the intron. 10-12 In addition to the core splicing signals, auxiliary splicing regulatory elements (SREs) have a critical role in recognition of exons and splicing efciency. 9,13,14 Furthermore, recent studies have indicated that pre-mRNA structure modulates the splicing machinery. 10,15,16 Previous studies suggest that disease-causing silent mutations distant to the core splice sites interfere with exon recognition either by affecting auxiliary SREs, leading to exon skipping, or by creating cryptic splice sites, resulting in partial deletion in exons. 9,17,18 Our current study, for the rst time in the literature of human genetic mutations, presents a synonymous variant outside the core splice sites that results in intron retention. Intron retention is a rare splicing defect that has been previously reported solely as a result of mutations residing in core 59ss or branch point motif sequences. 19-21 We had previously reported a translationally silent mutation c.7464C.T (p.Gly2488) in exon 44 of VWF, 25 bp after the 39ss and 85 bp downstream of the 59ss, in a patient with type 1 VWD. 22 In the present study, in vivo and ex vivo transcript analysis unexpectedly revealed that it inhibited splicing at the 59 donor splice site, despite its 85-bp distance to the 59ss, and led to an aberrant transcript with a retained intronic region in mature mRNA. The previously described mechanisms for exonic substitutions, such as contribution to cis- regulatory elements, could not provide a plausible explanation for this Submitted 12 February 2016; accepted 8 August 2016. Prepublished online as Blood First Edition paper, 19 August 2016; DOI 10.1182/blood-2016-02- 699686. *H.Y. and A.B. contributed equally to this study. The online version of this article contains a data supplement. There is an Inside Blood Commentary on this article in this issue. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. © 2016 by The American Society of Hematology 2144 BLOOD, 27 OCTOBER 2016 x VOLUME 128, NUMBER 17 For personal use only. on April 13, 2018. by guest www.bloodjournal.org From

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Page 1: splice site

Regular Article

THROMBOSIS AND HEMOSTASIS

Intron retention resulting from a silent mutation in the VWF gene thatstructurally influences the 59 splice siteHamideh Yadegari,* Arijit Biswas,* Mohammad Suhail Akhter, Julia Driesen, Vytautas Ivaskevicius, Natascha Marquardt, and

Johannes Oldenburg

Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany

Key Points

• This study demonstratesallosteric RNA structurealteration resulting from anexonic variation, therebyinterfering with splicing.

• This study details a novelmechanism by which silentmutation distant to the 59splice site could still result inintron retention.

Disease-associated silent mutations are considered to affect the accurate pre–

messenger RNA (mRNA) splicing either by influencing regulatory elements, leading to

exon skipping, or by creating a new cryptic splice site. This study describes a new

molecular pathologicalmechanismbywhich a silentmutation inhibits splicing and leads

to intron retention.We identified a heterozygous silentmutation, c.7464C>T, in exon44of

the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In

vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron

44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the

59 splice site (59ss). The abnormal transcript with the retained intronic region coded a

truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal

immunofluorescence characterizations of blood outgrowth endothelial cells derived from

thepatient confirmed thepresenceof the truncatedproteinbydemonstratingaccumulation

of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure

analysis revealed that this substitution, despite its distal position from the 59ss (85 bp

downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 59ss. This hairpin

sequesters the 59ss residues involved inU1 small nuclear RNA interactions, thereby inhibitingexcisionof the pre-mRNA intronic region.

This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by

structural changes in the pre-mRNA. (Blood. 2016;128(17):2144-2152)

Introduction

von Willebrand factor (VWF) is a multimeric plasma glycoproteinsynthesized in endothelial cells and platelet precursors and playscrucial roles in hemostasis.1 The VWF gene (VWF) is composed of 52exons spanning ;178 kb of the genome, and it is transcribed into an8.8-kb messenger RNA (mRNA).2 Deficient VWF results in vonWillebrand disease (VWD), which is classified as quantitative (type 1and type 3) or qualitative (type 2).3,4 Type 1 VWD, characterized bypartial reduction in VWF levels, is the most common form of thedisorder.5 Inheritance of type 1 VWD is considered to be autosomaldominant; however, in ;15% of index cases, more than a singlecandidate VWF variant is detected.6-8

Pre-mRNA splicing is regulated by consensus core splicing sitescomprising the 59 splice site (59ss), the 39 splice site (39ss), and thebranch point sequences.9 The splicing reaction is initiated by RNA-RNA base pairing of the consensus 59ss motif and the U1 smallnuclear RNA (snRNA) terminus, which is followed by excision ofthe 59 end of the intron, ligation of the adjacent exons, and releasing ofthe intron.10-12 In addition to the core splicing signals, auxiliarysplicing regulatory elements (SREs) have a critical role in recognitionof exons and splicing efficiency.9,13,14 Furthermore, recent studies

have indicated that pre-mRNA structure modulates the splicingmachinery.10,15,16

Previous studies suggest that disease-causing silent mutationsdistant to the core splice sites interfere with exon recognition either byaffecting auxiliary SREs, leading to exon skipping, or by creatingcryptic splice sites, resulting in partial deletion in exons.9,17,18 Ourcurrent study, for the first time in the literature of human geneticmutations, presents a synonymous variant outside the core splice sitesthat results in intron retention. Intron retention is a rare splicing defectthat has been previously reported solely as a result ofmutations residingin core 59ss or branch point motif sequences.19-21

We had previously reported a translationally silent mutationc.7464C.T (p.Gly2488) in exon 44 of VWF, 25 bp after the 39ss and85 bp downstream of the 59ss, in a patient with type 1 VWD.22 In thepresent study, in vivo and ex vivo transcript analysis unexpectedlyrevealed that it inhibited splicing at the 59 donor splice site, despite its85-bp distance to the 59ss, and led to an aberrant transcript with aretained intronic region in mature mRNA. The previously describedmechanisms for exonic substitutions, such as contribution to cis-regulatory elements, could not provide a plausible explanation for this

Submitted 12 February 2016; accepted 8 August 2016. Prepublished online as

Blood First Edition paper, 19 August 2016; DOI 10.1182/blood-2016-02-

699686.

*H.Y. and A.B. contributed equally to this study.

The online version of this article contains a data supplement.

There is an Inside Blood Commentary on this article in this issue.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked “advertisement” in accordance with 18 USC section 1734.

© 2016 by The American Society of Hematology

2144 BLOOD, 27 OCTOBER 2016 x VOLUME 128, NUMBER 17

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Page 2: splice site

event. Interestingly, a combination of in silico secondary and tertiarystructure analysis of thepre-mRNAclarified this long-distance influenceby demonstrating allosteric modifications in RNA folding resultingfrom the given mutation, which inhibited accessibility of the 59 coresplice site.

Materials and methods

Patients: phenotypic analysis

An 11-year-old girl index patient (IP) diagnosed with VWD from a non-consanguineous family and her parents were included in the study. Labo-ratory investigation of VWF antigen (VWF:Ag), VWF binding to plateletglycoprotein Ib, factor VIII coagulant activity, and VWF multimers(1.2% [w/v] and 1.6% [w/v] agarose gels) was performed as previouslydescribed.4,22 The bleeding score was calculated on the basis of a bleedingquestionnaire for type 1 VWD.23 This study was approved by the local ethicscommittee, and informed consent was obtained from all patients (vote 091/09).

Gene analysis

Genomic DNA was isolated from peripheral whole blood of the IP and herparents by standard methods. Mutation screening analysis was carried out bydirect sequencing of exons 1 to 52, including exon/intron boundaries, 59 and 39untranslated regions, and the promoter region of the VWF as described before.22

In addition, sequence analysis of thewhole intron44ofVWFwasperformed.Thecurrent single-nucleotide polymorphism (SNP) database was checked for thepresence of unknown substitutions through the National Center for Bio-technology Information (http://www.ncbi.nlm.nih.gov/SNP, accessed March2015). The splice-site prediction tool Human Splicing Finder version 2.4.1(http://www.umd.be/HSF, accessed February 2014) was used to analyze theeffect of the novel identified variant on splicing regulatory sites.24

The Multiplex Ligation-Dependent Probe Amplification assay (kits P011and P012; MRC-Holland, Amsterdam, The Netherlands) was applied to detectthe potential presence of VWF exon rearrangements.25

BOEC isolation

Blood outgrowth endothelial cells (BOECs) were isolated from blood of the IP,her mother, and 3 healthy individuals based on the published standardizedprotocols (see supplemental Methods, available on the BloodWeb site).26

RNA isolation and RT-PCR assay

The total RNAwas isolated fromwhole blood of the IP, her mother, and healthycontrol subjects using the Tempus SpinRNA IsolationKit (AppliedBiosystems,UnitedKingdom) according to themanufacturer’s instructions. In addition,RNAwas extracted from cultured BOECs and platelets by using RNeasy Mini Kit(QIAGEN, Germany). The isolated mRNA was reverse transcribed (RT) tocomplementary DNA (cDNA), and subsequently, VWF cDNAwas polymerasechain reaction (PCR)-amplified in 14 overlapping fragments containingmultipleexons using the QIAGEN LongRange 2Step RT-PCR Kit according to themanufacturer’s recommendations. PCR reactions were performed in thefollowing cycling conditions: 3 minutes at 93°C; followed by 35 cycles of15 seconds at 93°C, 30 seconds at 55°C, and 2 minutes at 68°C; and a finalextension of 2 minutes at 68°C. To allow amplification of the probable aberranttranscriptwith the larger size,RT-PCRsof overlapping segments 12 and13wererepeated using the same primers but increasing the extension time of PCRcycling from2minutes to 6, 8, and 10minutes. The sequence and position of theprimers and the product sizes of theRT-PCRare shown in supplemental Table 1.The RT-PCR products were separated on 1% agarose gel and sequenced toidentify the variations in the mRNA transcript.

Subsequent RT-PCR reactions using 4 allele-specific primer combinationswere performed to ascertain whether intron 44 was retained within the maturemRNA. In the first pair, a forward primer was designed across the junction ofexon 40-41, and a reverse primer was designed to target a sequence in intron 44.

In the second, third, and fourth primer pairs, forward primers targeted 3 differentsites in intron 44, and the reverse primer was directed at the exon 48-49 junction(supplemental Table 2).

VWF expression assessment and confocal

immunofluorescence (IF) microscopy of BOECs

VWF:Ag levels in themediumand lysate ofBOECsweremeasured as describedin supplemental Methods. Furthermore, western blotting of intracellular VWFwas performed to evaluate production of the precursor VWF (pre-pro-VWF) inBOECs (supplemental Methods).

In addition, BOECs were fixed and stained with immunofluorescentantibodies to visualize VWF, PECAM-1, cis- and trans-Golgi compartments,and endoplasmic reticulum (ER) (supplemental Methods). The comparativedegree of colocalization for the wild-type (wt) and mutant VWF was calculatedas mean Pearson correlation coefficient.27

In silico secondary and tertiary pre-mRNA structure analysis

Structural analysis of the pre-mRNA was performed to generate information onthe secondary and tertiary structure corresponding to the wt and mutant variant.Secondary structure analysis was performed on the mfold Web server (http://mfold.rit.albany.edu/?q5mfold, accessedFebruary 2014).28,29 Tertiary structureanalysis was performed on 2 ab initioWeb servers and on the basis of secondarystructure prediction made by mfold on RNAComposer (http://rnacomposer.cs.put.poznan.pl, accessed May 2014).30 The Rosetta online platform (http://rosie.rosettacommons.org/rna_denovo, accessed April 2014) was used to generate denovo models of RNA sequences 30 nucleotides long surrounding the mutatednucleotide for both themutant andwt sequences.31 Because the Rosetta RNAdenovo platform has a size limitation, in order to investigate any distal allostericinfluences on structure upstream or downstream of the mutation loci, wegenerated an ab initio model of a longer sequence (137 nucleotides) of pre-mRNA (including the mutated nucleotide and parts of exon 44 and intron 44covering both the 59ss and 39ss) on the iFold server (http://troll.med.unc.edu/ifold, accessed June 2014).32 In another type of model generation, the secondarystructure prediction for the lowest energy structure was downloaded in dot-bracket format from mfold and was used as an input file on the RNAComposerserver to generate the 3-dimensional coordinates corresponding to the second-ary structure prediction. Default modeling parameters were used for all Webservers. Modeling was also performed with all these servers for generating the3-dimensional structure for the U1 snRNA sequence (only for first 69 nucle-otides). Subsequently, in silico docking for the U1 snRNA structures was per-formed on the 3-dimensional structures of themutant andwt structures generatedfrom iFold on the ZDOCK server (version 2.3; http://zdock.umassmed.edu,accessed September 2014).33 Because the server applies Fanelli parameters toimplement protein-protein or protein–nucleic acid docking, theRNA-RNAdockwas analyzed only from a coarse-grained perspective.34 To compare theeffectiveness of individual base pairs in RNA-RNA interaction, a hypotheticalU1 snRNA–59 donor splice site complex structure was reproduced by firstcofolding the 2 RNAs on the RNAcofold server (http://rna.tbi.univie.ac.at/cgi-bin/RNAcofold.cgi, accessedSeptember2014) and subsequentlygeneratingthe 3-dimensional coordinates for the 2-dimensional prediction using theRNAComposer server. The tertiary structures were visualized and analyzed, andtheir images were rendered with YASARA version 13.8.26.35 SupplementalTable 3 defines the sequences submitted for structure prediction with respect tothe location of the mutation and the splice sites.

Results

Characterization of patients

The type 1 VWD IP with a cumulative bleeding score of 17 had ahistory of epistaxis, frequent bruises, prolonged bleeding from smallwounds, and oral-cavity bleeding. Furthermore, she had a tendency tobleed after tooth extraction and surgery. The IP had a significantlyreduced VWF:Ag of 9 IU/dL and a prolonged platelet function assay

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(PFA-100) closure time (Table 1). Her VWFmultimer profile revealednormalmultimer size distribution but a reduction in concentration of allmultimers due to her low VWF.Ag level (supplemental Figure 1). Onehour after desmopressin administration, the IP’s VWF:Ag level wasincreased from 9 to 19 IU/dL (alongwith an increase inVWF ristocetincofactor activity from,6 to 19 IU/dL). These levels remained almostpersistent 2 hours after treatment. However, this poor VWF responseto desmopressin was not clinically sufficient in many occasions, andtreatment with VWF/factor VIII concentrates was required.

Although both parents of the IP had no bleeding history, theyshowed prolonged closure time and a modestly decreased VWF:Aglevel, indicating that they have a risk factor for bleeding (Table 1).

VWF gene mutations

DNA sequencing revealed a heterozygous unknown synonymousvariant, c.7464C.T (p.Gly2488), in exon 44. Genetic analysis ofthe parents showed that the IP had inherited the unknown variant,c.7464C.T, from her mother. No other VWFmutations were detectedby the sequencing of all exons and the promoter region. No exonrearrangement was found in the explored region of VWF by MultiplexLigation-Dependent Probe Amplification analysis. The impact of thedetected silentmutation (c.7464C.T)on splice enhancing and silencingmotifs (ie, SREs) was evaluated with the Human Splicing Finderprediction tool, and the results are presented in supplemental Table 4.

In addition, no unknown variant was detected after sequenceanalysis of intron44; only8SNPs thatwere already recorded in theSNPdatabase were identified in intron 44 (supplemental Figure 2). Theminor allele of each detected SNP had a frequency of .10%(supplemental Figure 2).

RNA analysis

Further RNA analysis was performed for assessment of the patho-genicity of the variant c.7464C.T with respect to RNA splicing. Inaddition, complete sequencingof theVWF cDNAwasperformedbasedon the assumption that theremight be anothermutation deep in an intronthat could affect splicing of the second allele.

Therewere no differences in either the number or the size of theRT-PCRamplification products of IPVWF cDNAcomparedwithwt on theagarose gel (Figure 1A). Moreover, the sequence analysis of the wholeVWF cDNA showed no abnormality. However, surprisingly, analysisof the cDNA sequence of fragments 12 and 13 (both compromisingexon 44 with the heterozygous variant c.7464C.T) demonstrated themonoallelic presentation of the wt transcript, c.7464C, in the sequencechromatogram, indicating no normalmRNA transcript from themutantallele (Figure 1A). The presence of heterozygous exonic SNPs in theother RT-PCR fragments indicated that the mutant transcript is present

but cannot be amplified, probably due to its large size, leading topreferential amplificationof thewt transcript.RepetitionofRT-PCRofsegments 12 and 13with an increase in extension time of PCR cyclingconfirmed this presumption. The RT-PCR of segment 12 showed2 amplicons: an expected 920-bp segment corresponding to thenormal transcript, and an aberrantly spliced transcript of ;3200 bpthat, in addition to the normal transcript, had an insert of ;2200 bp(Figure 1B). Likewise, repetition of RT-PCR of fragment 13 confirmedan insert with same size (supplemental Figure 3). Further RT-PCRswith 4 pairs of allele-specific primers confirmed the retention ofwholeintron 44 (Figure 1C). However, the RT-PCR of the control RNAsperformed with the same allele-specific primers failed, as expected(Figure 1C).

The same aberrant transcript was observed when the IP’s plateletRNA (data not shown) and BOECRNA (supplemental Figure 4) wereused as the template. Similarly, the mother with the same substitution(c.7464C.T) detected in exon 44 of the VWF showed the same defectin RNA splicing (Figure 1B; supplemental Figure 3).

Confocal IF microscopy of BOECs

The isolated BOECs were characterized by immunofluorescentstaining for the endothelial cell-specific marker, PECAM-1, andVWF (Figure 2A). Confocal IF analysis showed that in contrast topervasive VWF staining in normal BOECs, VWF staining amongBOECs isolated from the IP was less and variable. Only 64 of 100inspected IP BOECs expressed VWF protein, which itself exhibitedsome degree of variability. Furthermore, VWF staining in the IPBOECs was mostly diffuse, with only a limited number of roundedWPBs compared with the VWF stored in discrete elongated WPBswithin normal BOECs (Figure 2A-B). Immunofluorescent VWFstaining of the mother-derived BOECs demonstrated a combination ofVWF stored in WPBs and diffuse staining (Figure 2A-B). Further IFanalysis illustrated that the diffuse VWF within the BOECs of the IPand her mother is colocalized with the ER marker, protein disulfideisomerase, demonstrating retention of truncated VWF proteins in theER (Figure 2B). Themean Pearson coefficient, representing the degreeof ER colocalization, was significantly higher (P, .01) for the BOECsof the IP and her mother (0.540 6 0.114 and 0.248 6 0.154,respectively) compared with normal BOECs (0.0406 0.019).

Expression of VWF in BOECs

To quantify total production of the VWF in BOECs, VWF:Ag levels(%) in secreted medium and lysates of the BOECs were determined.Data are presented as mean6 standard deviation. Secreted VWF fromthe IP-derived BOECswas considerably lower (P, .01) than healthy-donor BOECs (13% 6 2.8% vs 72% 6 36.6%). The significant

Table 1. Phenotypic characteristics and genetic data of the IP and her parents

VWD diagnosis Mutation Age, yBloodgroup

Closure time PFA-collagen/ADP, s

VWF:Ag,IU/dL

VWF:GPIb,IU/dL

FVIII:C,IU/dL Multimer

IP Type 1 VWD c.7464C.T 11 A1 .300 9 6 35 Normal multimer

size distribution*

Mother Has risk factor

for bleeding

c.7464C.T 45 A1 149 49 46 92 Normal

Father Has risk factor

for bleeding

No mutation 46 A1 126 44 35 108 Normal

Normal

range

— — — — 71-118 65-165 64-150 70-157 —

ADP, adenosine 59-diphosphate; FVIII:C, factor VIII coagulant activity; PFA, platelet function assay; VWF:Ag, VWF antigen; VFW:GPIb, VWF binding to platelet

glycoprotein Ib; —, not applicable.

*The size distribution of the multimer pattern was normal but the concentration of the all multimers was reduced.

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reduction in secretion of VWF was not associated with a subsequentincrease in intracellular accumulation. In fact, the amount of the VWFin lysates of the IP’s BOECs was below the detection limit (,3%)compared with a VWF:Ag level of 24% 6 9.5% detected in normalBOEC lysates. Furthermore, the amount of VWF in both medium andlysates of the mother’s BOECs was reduced (P, .05) compared withnormal BOECs (23%6 3.5% vs 72%6 36.6% and 10%6 5.4% vs24% 6 9.5%, respectively) but they were still higher than thosemeasured in the IP’s BOECs (P, .05) (Figure 2C).

Western blot analysis of the healthy-donor BOEC lysates (concen-trated 103) revealed two protein bands representing pre-pro-VWFandmature VWF (Figure 2D). However, no protein band was detectedafter electrophoresis of the same amount of the 103 IP BOEC lysates.Only weak protein bands were visualized when electrophoresis

and blotting of the IP BOEC lysates were repeated with moreconcentration (303).

Secondary and tertiary structure predictions of pre-mRNA

The 3-dimensional structure prediction using the ab initio servers/RNAComposer and the 2-dimensional structure prediction presented aunique summation/conclusion for the effects observed in the cDNAanalysis experiments. Although the predictions performed on differentservers differed from each other to variable degrees, a commonconclusion derived from all the servers was that the mutation bringsabout gross changes in the overall structure of the pre-mRNA. The abinitio structures generated from iFold differed completely between thewt and themutant sequences (Figure3A).Most remarkably, the59donorsplice-site region forms part of a highly ordered stem-loop or hairpin

VWF mRNA

12 3

11437bp

Intron 44

23

4

4

3000

Healthy Control

Index Patient

Mutant cDNA PCR product:3130 bp

Wild- type cDNA PCR product:920 bp

cDNA-wt allele: c.7464C

gDNA: c.7464C/T

cDNA-mutant allele: c.7464T

15001000

3000

1500

500

3000 bp

1000 bp

IP Mother

1M 2 3 4 5

Forward

Forward

Intron 44

Reverse

Reverse

Control

M 1 2 3 4 M

2000 bp-1500 bp-1000 bp-

1 2 3 4

Index Patient

1000

500

5 6 78

9 1011

12

1314

A

B

C

896 bp

1683 bp1873 bp

Figure 1. RT-PCR products on agarose gel. (A) Schematic scale of the coding region of VWF (exons 2-52) with the primer positions designed for amplification of the full-

length VWF mRNA and corresponding amplicon segments. Agarose gel electrophoresis image shows the 14 overlapping RT-PCR products of VWF using total RNA from the

IP’s blood as template, under thermocycling conditions with 2 minutes of extension time. The sequence chromatogram of segments 12 and 13 (both covering exon 44 carrying

the silent mutation c.7464C.T) demonstrate single-peak manifestation of wt nucleotide C, c.7464C, indicating a fail in amplification of the mutant transcript. Lanes 13 and 14

were run in a separate gel but with similar running conditions. (B) RT-PCR products of segment 12 amplified with primers residing in exon 39 and exons 45/46 boundary, and

with increased extension time (6 minutes) of thermocycling. RT-PCR products of RNA obtained from blood of the IP and her mother demonstrate a smaller product (920 bp)

relevant to the normal transcript and an aberrant larger fragment (3130 bp) corresponding to the retained intron 44 in mRNA, whereas RT-PCRs using RNA from 5 healthy

control subjects as template show only the smaller normal fragment (lanes 1-5). (C) RT-PCR amplification using allele-specific primers to confirm intron 44 retention. The

primer combinations and expected amplicon sizes, if intron 44 is retained, are as follows: segment 1 (1437 bp), forward primer in exons 40/41 boundary and reverse primer

targeted in intron 44, 861 nucleotides downstream of the exon 44 (lane 1); and segments 2, 3 and 4 (length 1873, 1683, and 896 bp, respectively), forward primers directed in

intron 44 in 3 different positions (1787, 1977, and 11764) and reverse primer in exons 48/49 boundary (lanes 2, 3, and 4). Sequence analysis of the cDNA segment 1

exhibited monoallelic presentation of the mutant variant T (c.7464T) in the sequence chromatogram, indicating that the aberrant transcript is derived solely from the mutant

allele. Lane M represents the molecular weight marker (1-kb ladder).

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structure in the mutated sequence (Figure 3B). All nucleotides that areknown to bind U1 snRNA are already sequestered by a combination ofcanonical and noncanonical base pairs within this hairpin, which is 42nucleotides long, stretching on to the end of the evaluated sequence(Figure 3Bii). In comparison, the wt sequence did not show any suchhairpins (Figure 3Bi). One canonical base pair between the 15G(59 donor splice-site residues) and114C, a noncanonical base pairingbetween18C (59 donor splice-site residues) and24G, and a backbonehydrogen bond between the phosphate group of12U (59 donor splice-site residues) and18C were all observed in the region that binds to U1snRNA in the wt sequence. Most other residues corresponding to U1snRNA binding were unfulfilled and, therefore, in single-stranded form(Figure 3Bi). In addition, the purine/pyrimidine base atoms that usuallyparticipate in hydrogen bonding were more exposed in the wt structureand, therefore, more amenable to binding to U1 snRNA (supplementalTable 5). Interestingly, the secondary structure prediction (iFold)(Figure 4), the 3-dimensional coordinate structures generated onRNAComposer (supplemental Figure 5), and theRosetta server structuresfor themutated andwt sequences also agreedwith the observationsmade

on the 3-dimensional ab initio structures, although the correlations werenot absolute. Similar to the observations made in the ab initio structures,many of the 59 donor splice-site residues that bind to U1 snRNA wereobserved to be free in the wt sequence but prebound or physicallyconstrained in a double-stranded form in themutated sequence.However,the base pairings suggested from the secondary structure predictions andthe ab initio structures varied significantly from one another.

The RNA-RNAdocking of U1 snRNA (supplemental Figure 6) andourpre-mRNAab initio structures (mutant andwt) showedan interestingconsequence for the mutation upon RNA-RNA interaction. The wtstructure U1 snRNA docking showed only 1 putative dock, which waslocalized proximal to the well-known U1 snRNA–59 donor splice-sitebinding region (Figure 3Ci). The mutated structure U1 snRNA dockingshowed multiple putative docks, but none within the vicinity of theU1 snRNA–59 donor splice-site binding region (Figure 3Cii). Thehypothetical U1 snRNA–59 donor splice-site complex showed inter-action of the wt 59ss consensus sequence 21 to 15 (UGUAGG) tonucleotides 4 to 9 of U1 snRNA. The nucleotides relevant to this kindof interaction have been illustrated in detail in Figure 5.

120

NormalBOECs

NormalBOECs

VWF PDI/ER MergeVWF PECAM-1 Merge

BOECs-IPBOECs-IP

BOECs-Mother

BOECs-Mother

Pre-Pro VWF

Mature VWF250 kDa

150 kDa

Healthy Donor10X

IP10X

IP30X

100

80

60

40

20

0Healthy Donors Index patient Mother

Secreted VWFIntracellular VWF

72 ± 36.6 13 ± 2.8 23 ± 3.524 ± 9.5 <3 10 ± 5.4

VWF:

Ag (%

)

A B

C D

Figure 2. Subcellular distribution and expression of VWF in the BOECs isolated from the IP and healthy donors. (A) Characteristics of the BOECs via staining of the

cell-specific markers VWF (light green) and PECAM (red) with secondary antibodies conjugated with Alexa Fluor-488 and Alexa Fluor-594, respectively. However, only 64 of

100 inspected IP BOECs emitted light green fluorescent signals, representing production of VWF protein. In the VWF-expressing IP BOECs, VWF staining is mostly diffuse,

accumulating around the nucleus of the cells, whereas in normal BOECs, VWF can be seen as distinct elongated structures, indicating storage in WPBs. The BOECs isolated

from the IP’s mother illustrate a combination of VWF stored in WPBs and diffuse staining. The white box points out secreting VWF strings in normal BOECs that are not visible

in the IP-derived BOECs. Bars represent 20 mm. (B) Diffuse staining observed in BOECs obtained from the IP and her mother (carrying the mutation c. 7464C.T) was

colocalized with protein disulfide isomerase (PDI). Staining was performed using primary antibodies anti-VWF (left channel, light green) and anti-PDI (ER marker; middle, red)

and secondary antibodies conjugated with Alexa Fluor-488 and Alexa Fluor-555, respectively. Colocalization of VWF and PDI staining is illustrated in the right channel

(Merge). Bars represent 10 mm. (C) Bar graph of the mean of VWF:Ag levels in the medium and lysates of BOECs obtained from the IP, her mother (3 independent

experiments, N 5 3), and 3 healthy donors (each 3 independent experiments, N 5 9). The mean VWF:Ag was determined after 103 concentration of the collected medium

and cell lysates of confluent BOECs in 75 cm2 flasks. Error bars indicate the standard deviation. (D) Western blot analysis of BOEC intracellular VWF after electrophoresis on

4% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Left lane shows both pre-pro-VWF and mature VWF in the lysate of the normal BOECs after 103

concentration. Middle and right lanes are representative of the IP’s BOEC lysates after 103 and 303 concentrations, respectively. WPBs, Weibel-Palade bodies.

2148 YADEGARI et al BLOOD, 27 OCTOBER 2016 x VOLUME 128, NUMBER 17

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Discussion

We identified a translationally silent variation in exon 44 of VWF,c.7464C.T, in a type 1 VWD patient with severely reduced VWF:Aglevels. Subsequent RT-PCR analysis proved that the given variant

impairs the efficiency of RNA splicing. Our gene analysis showed thatthe IP inherited this disease-causing silent mutation from her mother,with reduced VWF:Ag levels. A comparison of VWF:Ag levels of theIP (9%) with those of her mother (49%) indicates that the investigatedsilent mutation accounts for a reduction of only 50% of VWF:Aglevels in plasma. Furthermore, a significant reduction of total VWF

A B

C

(i) (ii) (i) (ii)-3A

-2G

+1G

+4G

+5G

+6U

+7C

+2U

+3A

-1U

(i) (ii)

Figure 3. Ab initio models of pre-mRNA and docking analysis. (A) Ab initio structures corresponding to wt (i) and mutated (ii) pre-mRNA sequences generated on iFold.

The structure is depicted in stick format and shown in cyan. The mutated residue location is depicted in blue and marked with a lavender-shaded area. Hydrogen bonds are

depicted as magenta dots throughout panels A-C. The backbone of the region corresponding to residues that bind to U1 snRNA is red, whereas the bases are yellow. This

region is also marked by the lavender-shaded area. (B) Region corresponding to U1 snRNA binding for the wt (i) and mutated (ii) pre-mRNA models at a closer view. Color

coding is as observed in panel A. The residues that bind to U1 snRNA are numbered in the mutated sequence structure model. In the wt sequence model, the unbound

residues corresponding to U1 snRNA binding are marked with lavender-shaded regions. (C) Coarse-grained depiction of the dock of U1 snRNA over the wt (i) and mutated (ii)

pre-mRNA sequence structure. Because the depiction is coarse-grained, the entire model is depicted only as a beaded trace. The trace is blue for the pre-mRNA sequence,

with only the region corresponding to U1 snRNA binding shown in red. The different putative U1 snRNA docked structures are colored differently. Because only 1 putative dock was

observed for the wt sequence, U1 snRNA is shown as light green in the dock between the U1 snRNA and wt pre-mRNA structures. The inset images provide a closer view of the

U1 snRNA binding region on the pre-mRNA structures. The proximal regions in the U1 snRNA and wt pre-mRNA structures are marked by the lavender-shaded area.

A B

Figure 4. Secondary structure prediction of pre-

mRNA sequence. Secondary structure of the wt pre-

mRNA sequence (A) and the mutated pre-mRNA

sequence (B) predicted by mfold. Many of the 59 donor

splice site residues (5 residues: 21U, 11G, 12U, 13A,

and 17C) that bind to U1 snRNA were observed to be

free in the wt sequence but prebound or physically

constrained in a double-stranded form in the mutated

sequence. The regions corresponding to residues that

bind to U1 snRNA are marked by the lavender-shaded

area. The mutated residue locations are also marked

by the magenta-shaded area and identified by a thick

arrow. The inset images in each panel correspond to a

close-up view of the region of pre-mRNA that binds to

U1 snRNA in the 3-dimensional model of the wt and

mutated structures generated from mfold secondary

structure prediction on the RNAComposer Web server.

The model structure is depicted in stick format (light

green). The residues that bind to U1 snRNA are shown

in blue. Hydrogen bonds are depicted as magenta dots.

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biosynthesis in the IP BOECs and the absence of a clear mature VWFband on western blot analysis of cell lysates imply that production ofVWF from both gene alleles is disrupted. Although genetic analysesand a full-length VWF mRNA investigation failed to detect any othermutation, a second causative variation inherited from the IP’s fatherimpairing gene transcription or mRNA stability could not be excluded.

In vivo and ex vivo transcript analysis demonstrated that thedetected single-nucleotide substitution variation localized 25 nucle-otides after the 39ss and 85 nucleotides downstream of the 59 donorsplice site interrupts the catalytic excision of intron 44 and leads tointron retention. The aberrant transcript with the intronic regioninsertion leads to premature termination after adding 50 amino acids;thiswould code a truncated protein that lacks the carboxy-terminal endof the VWF protein required for dimerization of VWF monomers inintracellular VWF processing.36,37 Confocal IF investigations con-firmed accumulation of a truncated protein in the ER of the IP’sBOECs, as expected.We therefore conclude that the truncated proteinsaccumulated in the ER will be degraded and result in quantitativedeficiencies in VWF. Intracellular retention of VWF is already knownas a common mechanism for type 1 VWD.6

The occurrence of intron retention rather than exon 44 skippingimplies that the given mutation does not have any impact on theaccuracy of splicing at 39ss. Therefore, it can be safely concluded thatalthough bioinformatics predictions suggest interferencewith cis-SREs(supplemental Table 4), this disease-causing silent mutation would not

exert its pathological influence through this mechanism. It is classicallyconsidered that disease-associated synonymousvariantsmight interferewith exon definition by affecting cis-regulatory elements and lead toexon skipping, as opposed to our aberrantly spliced transcripts inwhichwe observed intron retention. Therefore, we explored the possibilitythat the variant might have a long-distance/allosteric influence onRNA structure (more specifically, the 59ss), thereby resulting in intronretention. Using in silico RNA secondary and tertiary structuredetermination, we have now shown that this is, in fact, the case forour variant. All predicted models for the mutated and wt sequencessuggested a change in structure between the wt sequence and themutated sequence. The typical RNA duplex formation U1:59ss ismediated via canonical base pairing of theU1 snRNA59 terminus to the59ss consensus sequence, spanning the positions 23 to 16 (CAG/GURAGU).38,39 However, natural 59ss exhibit considerable variationin different positions that lead to alternative noncanonical base-pairingregisters, including bulged or shifted nucleotides.39-41 In keeping withthe variations in natural 59ss sequences, our hypothetical pre-mRNA–U1 snRNA duplex structure (generated on the RNAcofold server)showed that the residue positions between21 to15 of our pre-mRNAparticipate in interacting with U1 snRNA. All models (tertiary andsecondary) suggest that the mutated sequence generates a stable longhairpin at the 59ss that structurally constrains the residues at this sitefrom binding to U1 snRNA. This does not happen in the wt sequencesin which most residues are unbound and available for interaction with

Figure 5. Hypothetical U1 snRNA–pre-mRNA com-

plex structure. The main image represents the

secondary structure depiction of the hypothetical U1

snRNA–pre-mRNA complex derived from the RNAco-

fold server. The inset images show the secondary

structure and the tertiary structure generated from the

same secondary structure prediction in closer detail. In

the secondary structure (lower inset), the residues of

U1 snRNA are shown in dark green, whereas the

residues of the pre-mRNA participating in the in-

teraction with U1 snRNA are red. The canonical base

pairing is shown by lines, whereas noncanonical ones

are represented by a blue dot. The tertiary structure

(upper inset) is depicted in stick format. U1 snRNA is

shown in light green, whereas the pre-mRNA interact-

ing residues are red. The rest of the pre-mRNA is gray.

2150 YADEGARI et al BLOOD, 27 OCTOBER 2016 x VOLUME 128, NUMBER 17

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Page 8: splice site

U1 snRNA. Thisfindingwas further confirmed by dockingU1 snRNAwith the mutated and wt pre-mRNA structures.

In line with our finding, recent studies involving genome-wideRNA structure profiling demonstrated universal secondary structurefeatures in human transcripts, in which 59ss were found to be lessstructured.42,43 In addition, a genome-wide RNA structure profiling ofArabidopsis thaliana revealed native stable structures at the 59ss ofunspliced pre-mRNAs in alternative spliced transcripts, which suggeststhat secondary structure inhibits the first step of the splicing.42,44

Moreover, a previous survey showed that synthetic stable hairpinssequestering 59ss had a strong inhibitory effect on splicing.15

Although the role of RNA structure in regulation of splicing isunderstood, the impact of disease-associated single-nucleotide variants onpre-mRNA structure, which could putatively affect the efficiency of RNAsplicing, is not adequately studied, except for few human genes such assurvival motor neuron and tau genes.14,15 Nevertheless, studies such asthe one reported on genome variations associated with spinal muscularatrophy disease in the survival motor neuron gene have demonstrated theinfluence of exonicmutations located in vicinity of the 59ss on pre-mRNAfolding.15,45,46 Our study adds to these findings by suggesting that theexonic variations not only have a localized influence but they also canmediatedistantbutcischangesbybringingaboutallostericmodifications inpre-mRNAstructureandfold.This isclearlydemonstratedbyourvariation,which is located in an exon and closer to the 39ss but influences the 59ss.

In conclusion, this study revealed a novel pathomolecular mech-anismbywhich adisease-causing silentmutationexertsmajor allostericchanges in RNA folding, thereby impairing the splicing process. Ourstudy, therefore, highlights the importance of investigating the putativeimpact of silent/exonic variations on pre-mRNA structure.

Acknowledgments

The authors acknowledge Jan Voorberg for technical advice onculturing BOECs.

This work was supported by a research grant from GrifolsDeutschland GmbH (J.O. and H.Y.).

Authorship

Contribution: H.Y. designed the study, performed the experimentalwork, interpreted the data, and wrote the paper; A.B. designed andperformed the molecular modeling and structural analysis, per-formed the confocal image analysis, and wrote the paper; M.S.A.performed the western blotting; J.D. evaluated and interpreted VWFmultimers and reviewed and edited the manuscript; V.I. and N.M.contributed with patient data; and J.O. designed and supervised thestudy, evaluated phenotypic and genotypic data of patients, andreviewed and edited the manuscript.

Conflict-of-interest disclosure: The authors declare no competingfinancial interests.

ORCID profiles: H.Y., 0000-0002-2006-8497; A.B., 0000-0002-4103-5854; J.O., 0000-0003-2655-3503.

Correspondence: Johannes Oldenburg, Institute of ExperimentalHaematology and Transfusion Medicine, University Clinic Bonn,Sigmund-Freud-Str 25, 53105 Bonn, Germany; e-mail: [email protected].

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online August 19, 2016 originally publisheddoi:10.1182/blood-2016-02-699686

2016 128: 2144-2152  

Natascha Marquardt and Johannes OldenburgHamideh Yadegari, Arijit Biswas, Mohammad Suhail Akhter, Julia Driesen, Vytautas Ivaskevicius, 

splice site′structurally influences the 5Intron retention resulting from a silent mutation in the VWF gene that 

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