Genetic Diversity of SCN5a Gene

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Research ArticleGenetic Diversity ofSCN5A Gene and Its Possible Association

with the Concealed Form of Brugada Syndrome Development inPolish Group of Patients

Beata UziwbBo-gyczkowska,1 Grzegorz Gielerak,1 PaweBSiedlecki,2 and Beata Pajdk3,4

Department of Cardiology and Internal Diseases, Military Institute of Medicine, Szaserow Street , - Warsaw , Poland Department of Bioinformatics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, - Warsaw, Poland BioVectis/Kucharczyk E, Pawinskiego A, - Warsaw, Poland Electron Microscopy Platform, Mossakowski Medical Research Centre, Polish Academy of Sciences, - Warsaw, Poland

Correspondence should be addressed to Beata Uzibo-Zyczkowska; [email protected]

Received April ; Revised August ; Accepted August ; Published October

Academic Editor: Diego Franco

Copyright Beata Uzibo-Zyczkowska et al. Tis is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Brugada Syndrome (BS) is an inheritedchannelopathy associated with a high incidenceo suddencardiacdeath. Te paper presents

the discovery o new genetic variants oSCNA gene which might be associated with the development o a concealed orm oBrugada Syndrome. Te study involved a group o patients ( men) with suspected concealed orm o Brugada Syndrome.Pharmacological provocation with intravenous ajmaline administration was perormed. Six patients with positive test results weresubjected to molecular analysis oSCNAgene with MSSCP method. Additionally, MSSCP genotyping was perormed or samplesobtained rom the amily members with Brugada Syndrome, despite the act that they had negative ajmaline challenge test results.Genetic examinations o theSCNAgene at positive patients showed known polymorphisms, new single nucleotide point(SNP) variants located at exons, and new single nucleotide point variants located at introns. Among new SNPs localized inSCNAgene exons three SNPs affected the protein sequence.

1. Introduction

About % o sudden cardiac death (SCD) cases are caused

mainly by electrical heart diseases []. In the recent years, aspecial attention has been paid to one o them, the BrugadaSyndrome (BS). Te diagnosis o BS is based on ECG criteriaas well as on the clinical picture. ypical BS ECG changesoccur as a result o ion unction disorders in the heart,which are caused by genetic mutations and lead to impropercourse mainly o repolarization processes in cardiomyocytes.Electrocardiographic eatures o the syndrome are dynamicand ECG curve is periodically normal; typical BS char-acteristics disappear, which makes it difficult to diagnoseBS. Concealed orm o BS causes underestimation o thisdiseases occurrence requency and a great number o peopleremain undiagnosed. Specic pharmacological provocation

tests with class I medicine are critical in revealing concealedECG eatures o BS.

.. Genetic Background of the BS. Te rst gene to be linkedto BS is theSCNA, the gene that encodes the-subunit othe cardiac sodium channel gene []. Almost mutationsat the SCNA gene have been identied at the syndromepatients since [, ]. Numerous detected mutations havebeen studied at the unctional level []. Te mutations at theSCNAgene occur in approximately % to % o BrugadaSyndrome cases. A higher incidence oSCNA mutations hasbeen reported in amilial rather than in sporadic cases [].

Other gene loci on chromosome , which is close tobut distinct rom SCNA, have recently been linked to thesyndrome (pp) [] and GPD-L []. Tose mutations

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 462609, 13 pageshttp://dx.doi.org/10.1155/2014/462609
http://dx.doi.org/10.1155/2014/462609http://dx.doi.org/10.1155/2014/462609


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resulted in the loss o unction o the cardiac sodium channel.Other genes associated with BS were reported in the last ewyears andshownto encode the and subunits o the L-typecardiac calcium channel [].

TeSCNAgene remains the main gene linked to BS. Onote, negativeSCNAresults generally do not rule out causal

gene mutations. Currently, knowledge o a specic mutationmay not provide guidance in ormulating a diagnosis ordetermining a prognosis. Mutation screening o the SCNAgene in patients with BS may only support a clinical overt orsuspicious diagnosis.

In recent years, the genotyping oSCNAgene was morecorrelated to the prognostic value than to the diagnosis othe BS itsel. Some o the SCNAmutations were related to aworse clinical course [] and others to a better [] prognosiso the BS patients.

2. Materials and Methods

.. Patient Populations. Te study involved a group o Polish patients ( men) with suspected concealed BS basedon specic ECG and/or clinical criteria:

(i) complete and incomplete right bundle branch block(RBBB) in ECG,

(ii) suspected but nondiagnostic ECG (types and ),

(iii) history o sudden cardiac arrest (SCA),

(iv) unexplained syncopes,

(v) sudden cardiac death (SCD) amongst amily mem-bers under ,

(vi) amily history o BS.

Te protocol o the study has been approved by theCommission or Bioethics. Written inormed consent wasobtained rom all o the patients.

.. Te Ajmaline Challenge est. All patients wereperormedwith pharmacological provocation with intravenous ajmalineadministration dosed mg/kg body weight or min, in saeconditions during -lead -hour Holter ECG monitoring.

In patients with positive test results, molecular tests o theSCNAgene were perormed.

Molecular tests were perormed in the amily members opatients with BS, even i the pharmacological provocation testwas negative in these individuals.

Occurrence o type electrocardiographic patterns (cove-shaped S elevation in right precordial leads with J wave orS elevation o mm (mV) atits peak ollowed by a negative wave with little or no isoelectric interval in more than oneright precordial lead, VV) or conversion o type or to the diagnostic type pattern afer ajmaline administrationwas considered as a positive test result []. Occurrence o type or S segment elevation was considered as negative testresult.

.. DNA Analysis. Te genetic analysis was conducted incollaboration with Kucharczyk E/BioVectis Company (War-saw, Poland). Genomic DNA were analysed in patients

with positive result o ajmaline challenge test (one patientwith positive result o ajmaline challenge test did not agreeto be genotyped) and in amily member o patients withnegative result o ajmaline challenge test. Genomic DNAwere extracted rom peripheral blood leucocytes ( L orozen blood was used). Isolation was perormed according

to the manuacturers protocol (A&A Biotechnology, Poland).Regions most likely to contain genetic mutations at exonso the SCNA gene were covered by PCR amplicons,covering exons and partial intron sequences, as previouslydescribed []. Several pairs o primers were synthesizedto ampliy with PCR reaction exons , , and due totheir large sizes, named as a, b, a, and so orth. PCRprimers were designed to cover the ull coding sequence(exons), as well as partial ragments o anking noncodingragments (introns). Te PCR products were separated onagarose gel to examine their specicity and to normalise theDNA concentration. Next, PCR products were screenedby multitemperature single-strand conormation polymor-phism (MSSCP) [] method or the presence o a single-point mutation or a polymorphism. Te MSSCP conditionswere individually optimized or each PCR product. MSSCPwas perormed on to % polyacrylamide gel, .% Cat .x BE buffer. For some regions, glycerol was addedto polyacrylamide gel up to % w/v concentration. MSSCPanalysis was perormed using DNA PointerSystem in .xBE buffer. emperature prole o electrophoresis was C. Electrophoresis was perormed with W o elec-trical power. Beore applying samples onto the gel, mino preelectrophoresis ( W at C) was perormed. At thebeginning, samples were maintained or min at V orconcentration. Subsequently, MSSCP separation was made.Te PCR products that have altered MSSCP mobility wereollowed by Sanger method. ng DNA o PCR productswere used as a matrix or sequencing reaction. Both strandswere sequenced at PCR products that revealed a geneticalternation. Genetic alterations were identied using theBLAS (Basic Local Alignment Search ool) program and itsBLASN version as well as UCSC (University o CaliorniaSanta Cruz) Genome Bioinormatics and NCBI (NationalCenter or Biotechnology Inormation) databases o single-nucleotide polymorphisms (SNPs).

.. Functional Analysisof SCNA Variants. An in silico anal-

ysis was perormed to evaluate the putative unctional impacto the three identied variations (SY, SF, and KD).We used the Polymorphism phenotyping- (PolyPhen-)server [], which integrates sequence-based and structure-based eatures to predict amino acid substitution effectsusing a nave Bayes classier. An amino acid change wasclassied as probably damaging i its probability scorewas greater than . or as possibly damaging i thescore was between . and .. o assess the inuenceo putative unstructured regions, we used DISOPRED []sofware along with DOMPRED [] to predict possibledomain boundaries and disordered binding regions. Finally,we used Phyre [] or structural eature predictions, mainly


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: Distribution o the examined population depending oninclusion criteria.

Inclusion criteria Number o included

patients

RBBB in ECG (completeand incomplete)

patients (.%)(i) RBBB complete

patients (.%)(ii) RBBB incompletepatients (.%)

History o SCA patients (.%)

Unexplained syncopes patients (.%)

SCD amongst amilymembers under

patients (.%)

Family history o BrugadaSyndrome

patients (.%)

Suspected butnondiagnostic ECG (types and )

patients (.%)(i) ype patients(.%)(ii) ype patients

(.%)

transmembrane regions and secondary structure using threedifferent algorithms.

3. Results

.. Patient Demographics. Study inclusion criteria were metby patients ( women and men) (able ). Average ageo the group was 31.612.2 years, rom to years. Averageage or women was29.68 10.9years while, or men, it was32.812.9 years. Te majority o patients (.%) were under

.Echocardiography in all the included patients revealed no

signicant organic heart disease.

.. Clinical Characteristics of the Group with Positive Result ofPharmacological Provocation est. Pharmacological provo-cation test was carried out on the whole study group.No signicant undesirable effects were observed. None othe patients met the criteria o discontinuation prior thescheduled conclusion o the study.

Positive test resulttype S segment elevation(Figure (a)), which was considered as diagnostic o BS, wasobtained in individuals (.%). Te other patients

(.%) had negative provocation test results (Figure (b)).Te group o patients with type S segment elevation

diagnostic o BS ollowing ajmaline administration consistedo men (.%) and woman (.%). Average age o thisgroup was 36.5 15.2, rom to years. Te group opatients with negative test results included men (.%)and women (.%). Average age o this group was30.9 11.7, rom to years. No statistically signicantcorrelation between gender, age, or body mass and ajmalinetest was observed.

As regards the group o individuals with positiveprovocation test results, patients had history o SCA (men),among whom, in person, the diagnosed SCA mechanism

was ventricular brillation. Te SCA mechanism in thesecond individual remains unknown. Both patients wereimplanted a cardioverter-debrillator. Within the group othe other patients, initially considered as asymptomatic, months ollowing the provocation test, syncopes occurred in person (woman), which was an indication o implanting

a cardioverter-debrillator. Te other individuals haveremained asymptomatic during the observation period last-ing rom to months.

.. Results of MSSCP Analysis and DNA Sequencing ofthe SCNA Sodium Gene. Genetic examinations oSCNAgene showed known polymorphisms: rs (A>G,AA), rs (A>G, intron), rs (>C, AG),rs (A>G, exon, nontraslanted region), rs(>C, exon, nontraslanted region), and rs (A>G,EE). Tree o them were noted at regions o codingproteins, two at noncoding regions andone at intron. Numer-ous new genetic variants were detected: at nontraslanted

regions ( SNPs), at introns ( SNPs), and in the proteincoding regions ( SNPs) DNA sequence variants causedno change in the coded amino acid, whereas altered thecoded amino acid.

An example o MSSCP analysis o amplicones repre-senting exons number and o the SCNA gene or particular patients is presented in Figure . On the otherhand, Figure shows an example o patients derieved-amplicone sequence analysis, which was compared withreerence sequences (Figure ). All detected polymorphismswere urther analyzed in context o their localization and itsimpact on aa SCNA protein sequence.

.. Known Polymorphisms. Te rs polymorphism atexon o theSCNAgene was ound in patients related toeach other. Itinvolved an alteration o nucleotides in position (A>G) o reerence sequence; however, detected

variant did not alter the aa in protein sequence (AA), thushaving no impact on protein unction. Te patients withthis variant were a man (ather) and a woman (daughter),both asymptomatic. Te pharmacological provocation testwas positive in the man and negative in the woman.

On the other hand, in exon b o the SCNA gene, aknown rs polymorphism wasidentied in person. Itinvolved a nucleotide alteration in position (A>G)o reerence sequence. Te patient with this variant was a man

with asymptomatic BS diagnosed based on pharmacologicalprovocation. Tis genetic alteration caused no amino acidchanges in protein sequence (EE).

Genotyping exon o the SCNA gene revealed a knownrs polymorphism in individuals. It involved analteration in nucleotides in position (A>G) oreerence sequence and was located at intron. Te personswere asymptomatic men with positive pharmacologicalprovocation test and a woman related to one o the men(daughter) with a negative result.

At exon c o theSCNAgene a known poly-morphism was identied in patients. Tis genetic changeinvolved a nucleotide alteration in position (>C)


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(a) (b)

F : (a) -lead ECG rom a patient with positive test result (beore and afer test). Te conguration o the S segment elevation inleads V to V is a coved type. (b) -lead ECG rom a patient with negative test result (beore and afer test).

o reerence sequence and caused no change in the amino

acid sequence in the coded protein (DD). Clinically,there were asymptomatic individuals related to each other(ather and son); one had negative result o pharmacologicalprovocation (ather) whereas the second patient had type change in S segment typical o BS. Te other patients werenot related; one was a man with symptomatic BS and withhistory o SCA while the other was a man with asymptomaticBS.

Further, analysis o exon o the SCNAgene revealedthe presence o known rs polymorphism, which wasdetected in patients. It involved a nucleotide alteration in position (A>G) o reerence sequence. Tis geneticchange occurred in the nontraslanted part o the exon in

mRNA position. Te described genetic change was present inalmost every patient. Its presence was not observed only in aman with symptomatic BS and history o SCA.

Another known polymorphism is rs identiedin patients at exon k o the SCNA gene. It involvedan alteration in nucleotides in position (>C) oreerence sequence, in mRNA position, and was locatedin the nontraslanted region. Te polymorphism was notobserved only in individuals rom the analyzed group. Teywere men (brothers)one with symptomatic BS and theother with asymptomatic BS.

.. New Genetic Variants in Nontraslanted Regions at Exons.At exon o the SCNA gene, a new polymorphism thatinvolved an alteration in nucleotides in position(G>A) o reerence sequence in mRNA position wasobserved. Te change was connected with the region tran-scribed on mRNA but is not translated as a protein. Teperson with this genetic variant was a man with diagnosedsymptomatic BS (with history o SCA). Te polymorphismwas not observed in other patients.

Another new DNA sequence change was observed in patients who were related to each other. It was connectedwith the change in nucleotides in position (G>C)o reerence sequence ound at exon . Te genetic variant

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

Exon 2 Exon 7

F : MSSCP separation o exon and exon PCR products.Note that samples number and number at exon and samplenumber at exon have distinct electrophoretic proles suggestingthe presence o minor genetic variants.

was present in mRNA position and was related to a non-traslanted mRNA part. Te patients with this polymorphism(a woman and a man) were asymptomatic; pharmacologicalprovocation test was positive in the man and negative in thewoman.

A new DNA sequence change was also observed at exong o theSCNAgene in patients. It involved an alterationin nucleotides in position (G>A) o reerencesequence in mRNA position. Te genetic alteration waspresent in a nontraslanted part o the exon. Te individualsincluded men with symptomatic BS and history o SCA.Moreover, the group included the brother and the ather othe patient with history o SCA, one with a negative and theother with a positive result o pharmacological provocation

test. Te last person with this polymorphism was a man withasymptomatic BS.

Genotyping exon i o the SCNA gene revealed new polymorphisms localized in a nontraslanted part o theexon. Tese changes were ound in all the examined persons,among whom patients had all the genetic variants and patients had two new sequence changes: in (C>)and (C>) position o reerence sequence, whilepolymorphism in position (C>) was observed inall the patients.

Furthermore, analysis o exon l o the SCNA geneshowed a new sequence variant in position (C>G)o reerence sequence in mRNA position. Te change


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GGTCTGCCCACCCTGCTCTCTGTCCCTGGGCATAGAATCAGGCCCATTGTCTGTGTCTTC

GAGCTTCCCCACAGGCAACGTGAGGAGAGCCTGTGCCCAGAAGCAGGATGAGAAGATGGC

GAGCTTCCCCACAGGCAACGTGAGGAGAGCCTGTGCCCAGAACCAGGATGAGAAGATGGC

GGTCTGCCCACCCTGCTCTCTGTCCCTGGGCATAGAATCAGGCCCATTGTCTGTGTCTTC

ggtctgcccaccctgctctctgtccctgggcatagaatcaggcccattgtctgtgtcttc

cagcttccccacaggcaacgtgaggagagcctgtgcccagaa caggatgagaagatggc

AAACTTCCTATTACCTCGGGGCACCAGCAGCTTCCGCAGGTTCACACGGGAGTCCCTGGC

AGCCATCGAGAAGCGCATGGCAGAGAAGCAAGCCCGCGGCTCAACCACCTTGCAGGAGAG

CCGAGAGGGGCTGCCCGAGGAGGAGGCTCCCCGGCCCCAGCTGGACCTGCAGGCCTCCAA

CCGAGAGGGGCTGCCCGAGGAGGAGGCTCCCCGGCCCCAGCTGGACCTGCAGGCCTCCAA

AGCCATCGAGAAGCGCATGGCGGAGAAGCAAGCCCGCGGCTCAACCACCTTGCAGGAGAG

AAACTTCCTATTACCTCGGGGCACCAGCAGCTTCCGCAGGTTCACACGGGAGTCCCTGGC

aaacttcctattacctcggggcaccagcagcttccgcaggttcacacgggagtccctggc

agccatcgagaagcgcatggc gagaagcaagcccgcggctcaaccaccttgcaggagag

ccgagaggggctgcccgaggaggaggctccccggccccagctggacctgcaggcctccaa

AAAGCTGCCAGATCTCTATGGCAATCCACCCCAAGAGCTCATCGGAGAGCCCCTGGAGGA

AAAGCTGCCAGATCTCTATGGCAATCCACCCCAAGAGCTCATCGGAGAGCCCCTGGAGGA

aaagctgccagatctctatggcaatccaccccaagagctcatcggagagcccctggagga

CCTGGACCCCTTCTATAGCACCCAAAAGGTGACTACCACCCACCTCCAGCCCTGCCTACC

CCTGGACCCCTTCTATAGCACCCAAAAGGTGACTACCACCCACCTCCAGCCCTGCCTACC

cctggaccccttctatagcacccaaaaggtgactaccacccacctccagccctgcctacc

cctctgtgcaactccc

CTTCTGTGCAACTCCC

CTTCTGTGCAACTCCC

1 11

61 71 81 91 101 111

21 31 41 51

121

181 191 201 211 221 231

131 141 151 161 171

241 251 261 271 281 291

301 311 321 331 341 351

361 371 381 391 401 411

421 431 441 451 461 471

Exon 2 ref.

Exon 2 samp.

Exon 2 samp. 1

Exon 2 samp. 1

Exon 2 samp. 1

Consensus

Exon 2 ref.

Consensus

Exon 2 ref.

Consensus

Exon2 ref.

Consensus

Exon 2 samp. 1

Exon 2 samp. 1

Exon 2 samp. 1

Exon 2 samp. 1

Exon2 ref.

Consensus

Exon 2 ref.

Consensus

Exon 2 ref.

Consensus

Exon 2 ref.

Consensus

F : Sequence alignment oSCNexon reerence (W) and M amplicon detected in sample (pos. heterozygote A>G,exon, amino acid pos. , polymorphism rs; pos. heterozygote G>C, exon, pos. in mRNA, nontraslanted region). Whitecolor shows changed nucleotides.

was ound only in person. Clinically, the person with thispolymorphism was a man with asymptomatic BS.

.. New Polymorphisms in Protein CodingRegions Tat CauseNo Alteration in the Coded Amino Acid. As regards the groupo examined patients, unknown genetic variants were

observed in one patient at exon c o the SCNA gene inprotein coding regions; they caused no change in the codedamino acid; thus, we should consider them a polymorphicchange. Te two novel polymorphic variants were detectedin positions (C>, FF) and (C>,LL). In both cases, the changed nucleotide is in the rdcoded position, which may inuence the act that it causes noalteration in the amino acid sequence. Tese genetic variantswere observed in a patient with diagnosed BS and history oSCA who required implantation o cardioverter-debrillator.

.. New Sequence Changes in the Protein Coding Regions TatAlter the Coded Amino Acid. During genetic analysis o the

SCNA gene, presence o unknown genetic variants thataltered the coded amino acid was noticed in patients.

Te rst variant contained change in nucleotides in position (C>A) o reerence sequence and wasobserved in patient with negative result o pharmacologicalprovocation. Tis genetic change was observed in exon o

theSCNAgene. It altered serine amino acids into tyrosinein position o the coded protein (SY). Tis variant wasound in none o the other patients.

Another genetic variant detected in one patient involveda change in nucleotides in position (C>) oreerence sequence, which altered serine amino acids intophenylalanine in protein position (SF). New variantwas ound at exon o the analyzed gene. Te change wasobserved in a patient with asymptomatic BS and its presencewas conrmed neither in amily members o the patient norin the other examined patients.

Te last new sequence variant, which, according to theUCSC Genomi Bioinormatic database, is ound in the


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1 2015aa number

SCN5A protein

Detected aa

alterations (W/M)

321

S/Y

DNA SNP

519 974

S/F K/D

N C

ex8, ex12, ex17,

C>A C> G>

F : Schematic illustration o Nav., showing the location o the novel putative amino acid changes.

N-terminal

Extracellular

Cytoplasmic

133

S1

150

180

S2

160

193

S3

210

243

S4

220

247

S5 S6 S7 S8 S9 S10 S11 S12 S13

273

374

359

389

416

737

718

751

770

798

781

808

833

861

837

915

942Membrane

C-terminal

(a)

S321Y HumDiv

HumVar

HumDiv

HumVar

HumDiv

HumVar

S519F

K974D

0.00 0.20 0.40 0.60 0.80 1.00

0.00 0.20 0.40 0.60 0.80 1.00

0.00 0.20 0.40 0.60 0.80 1.00

0.00 0.20 0.40 0.60 0.80 1.00

0.00 0.20 0.40 0.60 0.80 1.00

0.00 0.20 0.40 0.60 0.80 1.00

Tis mutation is predicted to be possibly damaging with a score of 0.667 (sensitivity:0.86; specicity: 0.91)

Tis mutation is predicted to be possibly damaging with a score of 0.635 (sensitivity:0.80; specicity:0.84)

Tis mutation is predicted to be probably damaging with a score of 0.992 (sensitivity: 0.49; specicity: 0.95)

Tis mutation is predicted to be probably damaging with a score of 0.997 (sensitivity:0.41; specicity: 0.98)

Tis mutation is predicted to be possibly damaging with a score of 0.775 (sensitivity:0.85; specicity: 0.92)

Tis mutation is predicted to be possibly damaging with a score of 0.516 (sensitivity:0.82; specicity:0.81)

(b)

DUF3451

461668

Na trans assoc

9531215

Ion trans

1594121

0.8

0.6

0.4

0.2

0

0 200 400 600 800 1000 1200 1400

Amino acid position

Condencescore

(c)

F : (a) Prediction o transmembrane, extracellular in cytoplasmic regions in SCNa protein. Location o the three new variants isdepicted as orange dots. (b) Prediction o unctional effects o nonsynonymous mutations done in PolyPhen sofware. All three variantshave two scores rom HumDiv (genomic oriented) and HumVar (diagnostic oriented). (c) Prediction o intrinsically disordered regions inSCNa (AA) done by DISOPRED. Domain organisation shown with respect to disordered regions. Orange color marks putativebinding sites. High condence score indicated better chance o unstructured ragment.

protein coding region, was observed in patients. Tepolymorphism was observed at exon o the SCNAgeneand involved a change in amino acids in position(G>) o reerence sequence. Tis genetic change alteredlysine amino acids into aspartic acid in position o

the coded protein (KD). BS was diagnosed in o thepersons while the other individuals were amily mem-bers o patients with negative results o pharmacologicalprovocation. Schematic representation o detected changes isillustrated inFigure . o evaluate the possible inuence o


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new missense mutations on channel unction, bioinormaticsanalysis has been conducted.

.. In Silico Functional Analysis of SCNA Variants. Tethree variants identied are located at the cytoplasmic region

o the SCNA-encoded protein (Figure (a)). Conrmeddisease associated genetic variants can be ound in closeproximity to each one o the new variants, as well as sites oamino acid modications (e.g., arginine methylation site at and or a glycosylation site at ). Tis would hintthat the observed variants are locatedin important regions orprotein unction. o urther explore their possible unctionalimpact, we employed a well-known bioinormatics algorithmPolyPhen. Te tool indicated a high possibility o damagecaused by mutating KD with prediction score close to (the highest possible) (Figure (b)). For the two othermutations, possible damage was also reported, but with lesserprobability. We sought to conrm these predictions withmore structural insights. We used three different sofwaretools to establish whether these variations would occur inunstructured and putative domain regions or not. Indeed,SF is located in a large domain o unknown unction(DUF, PFAM: PF), which is also predicted as anunstructured/disorderd region by all three bioinormaticsmethods (Figure (c)). Tis would suggest a possible proteinbinding interaction in this region, which could be hamperedby this variant (especially since serine contains a hydroxylicpolar group and phenylalanine is hydrophobic and aromatic).On the other hand, SY is also located in transmembraneion channel amily domain (Ion trans, PF) which is predictedto be structured. Te same goes with KD, located just atthe beginning o the sodium ion transport-associated domain(Na trans assoc, PF). Again, this is a structured regionbut very close to the predicted unstructured binding region().

.. New Point Mutations Found at Introns. In the regionso theSCNAgene, which, according to the UCSC GenomeBioinormatics database, are at introns, new point muta-tions were ound.

MSSCP analysis o exon o the SCNA gene detected new mutations within the intron. Te rst was a mutationin position o reerence sequence and involveda type A insertion. Te second mutation was an alteration

in nucleotides in position (>A) o reerencesequence. Te two said genetic changes were conrmed in patients, among whom were asymptomatic and weresymptomatic ( with history o SCA and with syncopes).Another genetic change detected at this exon in the other individuals was a change in nucleotides in position(G>) o reerence sequence. Te rst patient had positiveprovocation result and history o SCA whereas the secondpatient was asymptomatic and also had positive provocationresult. Te last sequence change at this exon was oundonly in one patient in position (G>) o reerencesequence. Te patient was asymptomatic with negative resulto ajmaline test.

MSSCP analysis showed presence o new genetic vari-ants at exon o the SCNA gene within the intron in patients out o . Te rst involved a change in nucleotidesin position (C>G) o reerence sequence. Tesecond mutation was a change in position (C>G)o reerence sequence. Tese DNA variants were observed in

all the examined patients apart rom one individual who wasasymptomatic and had negative result o pharmacologicalprovocation test. At exon o the SCNA gene, a DNAsequence variant within the intron (pos. , C>G) wasidentied in patients out o with positive ajmaline provo-cation test. Moreover, patients rom this group had symp-tomatic BS and had an implanted cardioverter-debrillatoreither due to history o SCA or due to unexplained syncopes.Concurrently, this polymorphism was conrmed in neithero the patients with negative results o provocation test. Teresults o MSSCP genotyping o amplicons representingSCNA gene are summarized in able . Additionally, able contains the list o intronic alterations and exchanges innoncoding regions along with short stretches o sequencealignments(W > M).

4. Discussion

Te major gene related to BS is the SCNA gene. Despitethe great development in molecular studies, it is estimatedthat mutations in the SCNA gene cause only about % o BS cases []. Tese mutations are more commonin amilial cases o the disease than in sporadic ones [].Negative results o genetic studies do not exclude causalgene mutations. Neither diagnosis nor prognosis o BS canbe based on genetic test results. In the presented work, amolecular analysis o the wholeSCNAgene was carried outwith respect to patients with positive provocation test (apartrom person who ailed to give their inormed consent)as well as their amily members (st degree o kinship) whogave their inormed consent. Due to both low predicted BSincidence in the Polish population (lack o accurate data) anda considerably low percentage o the known genetic changesbeing the underlying cause o the disease (% as above),the work was limited only to analyzing the occurrence othe known mutations. Te molecular study o the exonsand short exon/intron ragments oSCN gene was carriedout including also the alterations in the sequence o the ewnoncodingregions o thegene (introns). In this study, thenew

genetic variants were ound both at exons and at introns. It isa commonly accepted act that the effects o DNA sequencechange depend on their location in the gene. However, all tooofen, it is assumed that only genetic alterations in the codingsequences, that is, at exons, have an impact on the clinicalcourse o the disease. Recent studies and ndings have shownthat intronic mutations may play a major role in the splicingprocess, alter its course, lead to coding sequence abnormal-ities, and consequently inuence the structure and unctiono the encoded proteins. Numerous data reported in scienticpapers show that both intronic and exonic alterations mayresultin an aberrantsplicingprocess,leading to the ormationo abnormal proteins, which, in turn, affects the severity o


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:TeresultsofMSSCP

genotypingofampliconsrepresentingSCNAgene.

Majorclinicald

ata

GeneticalterationsinDNAsequencesofSCNamplicons

(referencese

quenceref|N.|Hs)

Patient

Asymptomatic

Negativepharm

acological

provocationtes

t

Fatherofthepatientwith

diagnosedBS

Amplicon:pos.,insertionA,intron

pos.homozygoteA,intron

Amplicon:

pos.insertionA,intron

pos.C>A,aminoacidpos.,S>Y

Amplicon.a:pos.G>,aminoacidpos.,K>D

Amplicon

c:pos.>C,exon,aminoacid

pos.D>D,rs

Amplicon

f:pos.A>G,exon,mRNAposition,nearUR,nontraslantedregion,rs

Amplicon

g:pos.heterozygoteG>A,mRNAposition,nearUR,nontraslantedregion

Amplicon

i:pos.C>,exon,mRNAposition,nearUR,nontraslantedregion

pos.C>,exon,mRNAposition,nearUR,nontraslantedregion

Amplicon

k:pos.>C,exon,mRNApos.,

UR,nontraslantedregion,polym

orphismrs

Patient

Historyofsudd

encardiac

arrest

Positivepharmacological

provocationtes

t

DiagnosedBS


pos.homozygote>A,intron

Amplicon:pos.G>C,intron

pos.homozygoteG>C,intron

Amplicone

:pos.C>G,intron

Amplicon

g:pos.G>A,exon,mRNAposition,nearUR,nontraslantedregio

n

Amplicon

i:pos.G>A,exon,mRNAposition,nearUR,nontraslantedregio

n

Patient

Historyofsudd

encardiac

arrest


provocationtes

t

DiagnosedBS

Amplicon:

pos.G>A,mRNApos.,nontraslantedregion

Amplicon:pos.G>,intron

Amplicon:pos.homozygoteC>G,intron

pos.homozygoteC>G,intron

Amplicon:

pos.C>G,intron

Amplicon

a:pos.G>,aminoacidpos.,K>D

Amplicon

c:pos.C>,exon,aminoacidpos.,I>I

pos.C>,exon,aminoacidposition,F>F

pos.>C,exon,aminoacidpos.,D>D,rs

Amplicon


Amplicon

g:pos.heterozygoteG/A,mRNA

position,nearUR,nontraslantedregion

Amplicon

i:pos.C>,exon,mRNAposition,nearUR,nontraslantedregion

pos.C>,exon,mRNAposition,nearUR,nontraslantedregion

Amplicon



orphismrs


9/14


10/14


:Continued.

Majorclinicald

ata

GeneticalterationsinDNAsequencesofSCNamplicons

(referencese

quenceref|N.|Hs)

Patient

Unexplainedsy

ncopes


provocationtes

t

DiagnosedBS

Amplicon:

pos.,A>G,aminoacidpos.,A

>A,polymorphismrs

pos.,G>C,exon,mRNApos.,nontraslantedregion



Amplicon:pos.C>G,intron


Amplicon

a:pos.G>,aminoacidpos.,K>D

Amplicon

:pos.heterozygoteA/G,intron,

polymorphismrs

Amplicon


Amplicon

i:pos.deletionC,exon,mRNA

pos.,nearUR,nontraslantedregion

pos.insertionA,exon,mRNApo

s.,nearUR,nontraslantedregion

pos.C>,exon,mRNApos.

,nearUR,nontraslantedregion

pos.C>,exon,mRNApos.,nearUR,nontraslantedregion

Amplicon



orphismrs

Patient

Asymptomatic


provocationtes

t



Amplicon:pos.C>G,intron


Amplicon:

pos.C>G,intron

Amplicon


Amplicon

i:pos.deletionC,exon,mRNA

pos.,nearUR,nontraslantedregion

pos.insertionA,exon,mRNApo

s.,nearUR,nontraslantedregion

pos.C>,exon,mRNApos.

,nearUR,nontraslantedregion

po

s.C>,exon,mRNApos.,nearUR,nontraslantedregion

Amplicon



orphismrs

Amplicon

l:pos.homozygoteC>G,exon,

mRNApos.,nearUR,nontraslan

tedregion


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: Te list o intronic alterations and exchanges in noncoding regions along with short stretches o sequences alignments (W>M).

Amplicon PositionDetected

polymorphism(W>M)

Localization Partial alignment (W>M)

Pos. G>AExon,

nontraslantedregion

CGCAGGCTCAGCGGCC

CGCAGGCTCAACGGCC

cgcaggctca cggcc

Amp.

Amp.

1 W

1 M

Consensus

1 11

Pos. G>CExon,

nontraslantedregion

CCAGAAGCAGGATGAG

CCAGAACCAGGATGAG

ccagaa caggatgag

2 W

2 M

Consensus

1 11

Amp.

Amp.

Pos.

Pos. Insertion A

>A Intron

GGAAGGGGG-CTTGTG

GGAAGGGGGACTTGAG

ggaaggggg cttg g

4 W

4 M

Consensus

1 11

Amp.

Amp.

Pos. G> IntronAGCACTGGCCTGGCAGT

AGCACTTGCCTGGCAGT

agcact gcctggcagtConsensus

1 11

4 W

4 M

Amp.

Amp.

Pos. G> IntronTGGTAGCACTGGCCTGG

TGGTAGCACTGTCCTGG

tggtagcactg cctggConsensus

1 11

W M

Amp. 4

Amp. 4

Pos.

Pos. G>CG/C

IntronCCTCTGACTGTGTGTC

CCTCTCACTGTCTGTC

cctct actgt tgtc

1 11

6 W

6 M

Consensus

Amp.

Amp.

Pos. C>G Intron GAACAAGCACGGGGTCGAACAAGGACGGGGTC

gaacaag acggggtc

1 11

7W

7 M

Consensus

Amp.Amp.

Pos. Insertion A IntronCTGGGTA-TGTGGCA

CTGGGTAATGTGGCA

ctgggta tgtggca

1 11

8 W

8 M

Consensus

Amp.

Amp.

Pos. A> IntronGCCAGTGGCACAAAAG

GCCAGTGGCTCAAAAG

gccagtggc caaaagConsensus

1 11

12 W

12 M

Amp.

Amp.

Pos. C> Intron

CCATTTCTACTTTG

CCATTTTTACTTTG

ccattt tactttg

1 11

Consensus

22 W

22 M

Amp.

Amp.

Pos. A>G IntronGCCAAGCAACCAGG

GCCAAGCAGCCAGG

gccaagca ccagg

1 11

Consensus

24 W

24 M

Amp.

Amp.

Pos.

Pos. Deletion

>A Intron

CCTGCTGAGCACTTTC

CCAGC-GAGCACTTTC

cc gc gagcactttc

1 11

Consensus

27 W

27 M

Amp.

Amp.


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: Continued.

Amplicon PositionDetected

polymorphism(W>M)

Localization Partial alignment (W>M)

Pos. A>G

Exon, nearUR,

nontraslantedregion

GGCCTCAGCCCC

GGCCTCGGCCCCggcctc gcccc

1 11

Consensus

28f W

28f M

Amp.

Amp.

g Pos. G>A

Exon, nearUR,

nontraslantedregion

CAAAGCAGAAGTGGAA

CAAAGCAAAAGTGGAA

caaagca aagtggaa

1 11

Consensus

28g W

28g M

Amp.

Amp.

i

Pos.

Pos. Pos. Pos.

Deletion C

insertion AC>C>

Exon, near

UR,nontraslanted

region

ATCGGAAGAGAG

AT-GGAAGAGAG

at ggaagagag

1 11

Consensus

28i W

28i M

Amp.

Amp.

CCCAGCCAGCCA-

CCCAGCCAGCCAA

cccagccagcca

1 11

Consensus

28i W28i M

Amp.Amp.

CCTTTTCTTCCCCTCCTG

CCTTTTTTTCCCCTCCTG

cctttt ttcccctcctg

1 11

Consensus

28i W

28i M

Amp.

Amp.

GGCCCCCTATTGTCTCCA

GGCCCCCTATTGTTTCCA

ggccccctattgt tcca

1 11

Consensus

28i W28i M

Amp.Amp.

k Pos. >CUR,

nontraslantedregion

TCTCCCATGGAGC

TCTCCCACGGAGC

tctccca ggagc

1 11

Consensus

28k W

28k M

Amp.

Amp.

l Pos. C>G

Exon, nearUR,

nontraslantedregion

CAGCGACATTTCTC

CAGCGAGATTTCTC

cagcga atttctc

1 11

Consensus

28l W

28l M

Amp.

Amp.

the disease symptoms. Tese mutations/polymorphisms atintrons leading to the disturbances o the splicing process aredescribed in the disorders o cardiovascular system [].

In the course o DNA analysis o the SCNA sodiumgene, the ollowing known polymorphisms were identied:rs, rs, rs, rs, rs, andrs. In this group, polymorphisms were observed in

the protein coding regions, in the nontraslanted regions,and one at the intron. None o them had been associated withBS beore. Also, new genetic variants were ound at exons inthe nontraslanted regions, at introns, and in the proteincoding regions that cause no change in the coded amino acid.None o point mutations (SY, SF, and KD) in theprotein coding regions that alter the coded amino acid hasbeen associated previously with BS []. According to Zimmerand Surber, as well as bioinormatic analysis, we are able tolocalize their positions in protein sequence. SY is localizedin the intracellular loop III, SF in the intracellular loop IV,whereas KD is localized in the C-terminal intracellularragment o SCNa protein. According to the bioinormatic

results, KD aa alteration is recognized as highly damagingor protein unction (prediction score amounted to about, the highest possible). wo other aa changes were alsoreported as possible damage; however, their probability scoreamounted rom . to ..

As mentioned previously, also intronic changes couldaffect protein unction. We perormed some basic bioinor-

matic analyses o detected changes; however, we obtainedcontradictory data. Due to the large number o detectedpolymorphisms in introns, we decided to perorm moredetailed analyses, includingin vitrostudies.

Te majority o detected polymorphisms and geneticchanges ound in the study had never been reported asmutations leading to development o BS. Te lack o datain the literature and the lack o a population control orthis part o the SCNA gene made it impossible to stateclearly whether the BS syndrome was signicantly associatedwith the mentioned changes or not. It is also noteworthythat several genes are associated with BS syndrome; thus,urther genetic study is needed. However, at least new


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polymorphisms/mutations that were ound in our patients oa specic phenotype are worth considering.

Special attention ought to be paid to genetic changespresent only in symptomatic patients, or example, withhistory o SCA. Tese genetic changes include the ollowing:

(i) a new polymorphism which involves an alteration innucleotides in position (G>A) o reerencesequence in mRNA position and developed in aman with history o SCA and BS diagnosed on thebasis o provocation test result; it was ound in noneo the other patients;

(ii) a new genetic variant at exon which involves analteration in nucleotides in position (G>A)o reerence sequence in mRNA position anddeveloped in patients including men with positiveprovocation test results and history o SCA as in otherindividuals (i.e., the brother and the ather o one othese men);

(iii) two new genetic variants at exon c in the proteincoding regions with no alteration in the coded aminoacid (C>, II; C>, FF) both developed in aman with historyo SCAand positive provocation testresults;

(iv) a new polymorphism at exon which involves analteration in nucleotides in position (C>G)o reerence sequence and developed in patients outo individuals with positive ajmaline provocationtest, o these patients had symptomatic BS ollowingimplantation o cardioverter-debrillator either dueto history o SCA or due to unexplained syncopes;

concurrently the mutation was conrmed in none othe patients with negative provocation test result.

Considering new data on the role o genetic changes notonly in BS diagnostics but also in prognosis or diagnosedpatients [, ], urther studies aimed at determining therole o the identied genetic disorders seem to be extremelyinteresting.

5. Conclusions

New genetic variants/polymorphisms in the SCNA geneare present in patients with concealed orms o Brugada

Syndrome, yet their role in pathogenesis requires urtherstudies.

Conflict of Interests

Te authors do not report any conict o interests regardingthe publication o this paper.

References

[] S. G. Priori, E. Aliot, C. Blomstrom-Lundqvist et al., askorceon sudden cardiac death o the EuropeanSociety o Cardiology,European Heart Journal, vol. , pp. , .

[] Q. Chen, G. E. Kirsch, D. Zhang et al., Genetic basis andmolecular mechanism or idiopathic ventricular brillation,Nature, vol. , no. , pp. , .

[] http://www.sm.it/uo-int.php?id=.

[] J. D. Kapplinger, D. J. ester, M. Alders et al., An internationalcompendium o mutations in the SCNA-encoded cardiacsodium channel in patients reerred or Brugada syndromegenetic testing,Heart Rhythm, vol. , no. , pp. , .

[] . Zimmer and R. Surber, SCNA channelopathiesan updateon mutations and mechanisms, Progress in Biophysics andMolecular Biology, vol. , no. -, pp. , .

[] E. Schulze-Bahr, L. Eckardt, G. Breithardt et al., Sodium chan-nel gene (SCNA) mutations in index patients with Brugadasyndrome:different incidences in amilial andsporadicdisease,Human Mutation, vol. , no. , pp. , .

[] B. London, M. Michalec, H. Mehdi et al., Mutation in glycerol--phosphate dehydrogenase -like gene (GPD-L) decreasescardiac Na+ current and causes inherited arrhythmias, Circu-lation, vol. , no. , pp. , .

[] C. Antzelevitch, G. D. Pollevick, J. M. Cordeiro et al., Loss-o-unction mutations in the cardiac calcium channel underlie anew clinical entity characterized by S-segment elevation, shortQ intervals, and sudden cardiac death, Circulation, vol. ,no. , pp. , .

[] B. Benito, O. Campuzano, R. Ishacet al., Role o genetic testingin risk stratication o Brugada syndrome,Heart Rhythm, vol., no. , supplement, article S, .

[] E. Lizotte, M. J. Junttila, M. P. Dube et al., Genetic modulationo brugada syndrome by a common polymorphism,Journal ofCardiovascular Electrophysiology, vol. , no. , pp. ,.

[] Q. Wang, Z. Li, J. Shen, and M. . Keating, Genomic organi-zation o the humanSCNAgene encoding the cardiac sodiumchannel,Genomics, vol. , no. , pp. , .

[] R. Kaczanowski, L. rzeciak, and K. Kucharczyk, Multitemper-aturesingle-strandconormation polymorphism, Electrophore-sis, vol. , pp. , .

[] I. A. Adzhubei, S. Schmidt, L. Peshkin et al., A method andserver or predicting damaging missense mutations, NatureMethods, vol. , no. , pp. , .

[] J. J. Ward, J. S. Sodhi, L. J. McGuffin, B. F. Buxton, and D. .Jones, Prediction and unctional analysis o native disorder inproteins rom the three kingdoms o lie,Journal of MolecularBiology, vol. , no. , pp. , .

[] D. W. A. Buchan, S. M. Ward, A. E. Lobley, . C. O. Nugent,K. Bryson, and D. . Jones, Protein annotation and modellingservers at University College London,Nucleic Acids Research,vol. , no. , pp. WW, .

[] L. A. Kelleyand M. J.E. Sternberg, Protein structurepredictionon the Web: a case study using the Phyre server, NatureProtocols, vol. , no. , pp. , .

[] C. Antzelevitch, Brugada syndrome,Pacing and Clinical Elec-trophysiology, vol. , no. , pp. , .

[] E. Schulze-Bahr, L. Eckardt, G. Breithardt et al., Sodiumchannel gene(SCNA) mutations in indexpatients withBru-gada syndrome: different incidences in amilial and sporadicdisease.,Human mutation, vol. , no. , pp. , .

[] M. Vatta, Intronic variants and splicing errors in cardiovascu-lar diseases,Heart Rhythm, vol. , no. , pp. , .

[] B. Benito, O. Campuzano, R. Ishacet al., Role o genetic testingin risk stratication o Brugada syndrome,Heart Rhythm, vol., supplement , p. S, .


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Genetic Diversity of SCN5a Gene

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