WO2016130088A1

WO2016130088A1 - Identification of novel single polymorphisms in kcnh2 gene and uses thereof

Info

Publication number: WO2016130088A1
Application number: PCT/SG2016/050069
Authority: WO
Inventors: Ashish Mehta; Winston Se Ngie Shim; Chrishan Julian Alles RAMACHANDRA; Philip En Hou Wong; Teng Hong TAN
Original assignee: Singapore Health Services Pte Ltd
Current assignee: Singapore Health Services Pte Ltd
Priority date: 2015-02-11
Filing date: 2016-02-11
Publication date: 2016-08-18
Anticipated expiration: 2017-08-11

Abstract

The present invention relates to novel single polymorphisms in the KCNH2 and their applications in molecular profiling of long QT syndrome 2.

Description

Identification of novel single polymorphisms in KCNH2 gene and uses thereof Field of the invention

The present invention relates to the field of molecular biology, in particular to the molecular profiling a condition and/or disease. Background of the invention

Genetic variants within DNA sequence found in the introns and exons are associated with predisposition towards manifestation of disease in relation to susceptibility and expressivity of symptoms. Single nucleotide polymorphisms (SNPs; DNA sequence variation when a single nucleotide in the genome sequence is altered) have been evaluated extensively in relation to cardiovascular diseases (CVD). The 2 classic complementary approaches used for relating genetic sequence variation to CVD risk are the linkage and association approaches.

Linkage approach investigates families with a whole genome scan consisting of hundreds of anonymous markers to identify genetic loci that may be related to disease susceptibility. Such linkage strategy identifies a segment of the genome (typically involving millions of bases of DNA) that segregates with disease. Fine mapping within these segments may lead to the identification of gene/s or elements that are disease causing. On the other hand, association strategy evaluates the relation of genetic variants, typically in unrelated individuals, to the presence versus absence of disease or to variation in values of a quantitative trait. The scientific rationale behind such association studies is that common genetic variants with modest effects contribute to the variation of complex disease in the population that may explain disease penetrance and expressivity. The recognition that groups of neighbouring polymorphisms in the genome are highly correlated has led to the concept of tag SNPs, which can be used as proxies for most of the common genetic variants in a region of linkage dysequilibrium. Tag SNPs are expected to greatly facilitate association studies because fewer markers are needed to be genotyped. Both linkage and association studies have been and continued to be providing valuable insights into genetic markers in the context of pathogenesis of CVD. Sinus rhythm is determined by complex interlinked genetic traits that govern electro-mechanical coupling of the myocardium. Such cardiac rhythm is in part tightly linked to the depolarization and repolarization of cellular membrane of cardiomyocytes, which in turn determine the action potential duration underlying QT interval of electrocardiogram (ECG). The overall action potential is governed by an interplay of key ion channels in cardiomyocytes. Among the major ion channels, hERG (human Ether-a-go-go-Related Gene; also known as KCNH2) channel is one of the most critical in determining repolarization of cardiomyocytes. It is therefore unsurprising that dysregulation of hERG channel precipitates sufferers to heightened risk of arrhythmia, syncope and sudden cardiac death. Mutations associated with the hERG channel pre-dispose patients to genetic long QT syndrome 2 (LQTS2). A variety of physiological stimuli (audio, emotion, hormone, exertion etc) or environmental triggers (diet change, electrolyte imbalance) could precipitate arrhythmias in patients in whom may not be clinically manifesting any pathological condition otherwise. LQTS2 arising from mutations in hERG channel encoded by KCNH2 gene constitutes an approximately 45% of clinical reported cases among all known subtypes of LQTS. Currently, there are more than 500 different mutations of the KCNH2 gene reported in the literature that may cause LQTS2 phenotype. It has previously shown that cardiomyocytes derived through induced pluripotent stem cells from LQTS2-specific patients mimic patient phenotype in a laboratory dish (Mehta et al 2014). Moreover, these cardiomyocytes also demonstrate arrhythmogenic episodes, spontaneously as well as in response to pharmacological challenges.

Diagnosis and management of long QT syndrome is complex (Vincent 2002; Schwartz and Crotti, 2011 ). In a clinical setup, LQTS2 patients are generally identified based on long QTc interval in their ECG followed by a genetic screening to identify the gene mutation. However, prolonged QTc interval alone does not discriminate between asymptomatic and symptomatic cases, which renders disease management challenging. Typically, symptomatic cases present clinical manifestations such as arrhythmogenic episodes and/or syncope but it would be substantially difficult to categorize asymptomatic cases due to the lack of overt clinical symptoms. In addition, the prognosis of asymptomatic cases is less well defined and prophylactic management is more complicated. For example, current symptom-free next-of-kin of the symptomatic patients may be at risk in future, yet currently there are no reliable methods to risk stratify such asymptomatic patients of their vulnerability to disease manifestation. Indeed, such asymptomatic patients form the bulk of patients being risk managed in today's healthcare system that takes up significant available medical resources.lt is desirable to understand why subjects with a prolonged QTc interval are asymptomatic and do not manifest clinical symptoms and also identify asymptomatic subjects for proper management. Summary of the invention

According to a first aspect, the present invention provides a method for predicting and/or diagnosing long QT syndrome 2 (LQTS2) comprising identifying a subject having one or more SNP variants selected from the group: a heterozygous CT or a homozygous TT for rs3807375, a heterozygous CT or a homozygous TT for rs2968857, a heterozygous GA or a homozygous AA for rs34657537, a heterozygous AG or a homozygous GG for rs12668582, a heterozygous TC or a homozygous CC for rs4725385, a heterozygous GA or a homozygous AA for rs2269001 , a heterozygous GA or a homozygous AA for rs1805120, a heterozygous CT or a homozygous TT for rs188262457, a heterozygous TC or a homozygous CC for rs1805121 , a heterozygous AG or a homozygous GG for rs1137617; and a heterozygous GA, GC, AC or a homozygous CC or ΔΔ for rs531460655; wherein Δ comprises a deletion; as being at risk of or suffering from LQTS2.

According to a second aspect, the present invention provides a method for genotyping and thereby classifying a subject comprising: Identifying (A) a subject having: a homozygous CC for SNP rs372582297; and a homozygous TT for rs11766022; or (B) a subject having (i) a heterozygous CG or a homozygous GG for rs372582297 and/or a heterozygous TG or a homozygous GG for rs372582297; and (ii) one or more variant SNPs selected from the group consisting of: a heterozygous CG or a homozygous GG for rs35277497, a heterozygous CT or a homozygous TT for rs1036145, a heterozygous GA or a homozygous AA for rs11763131 , a heterozygous AT or a homozygous TT for rs 3244889; as a healthy subject without LQTS2 or an asymptomatic LQTS2 subject

Brief description of the figures Figure 1 : Typical multi-electrode array tracings showing extracellular field potential recordings of normal (control) and LQTS2 hiPSC-CM clusters. Note prolonged field potential duration (FPD) in LQTS2 hiPSC-CMs as compared to controls. B-C, Box plot showing rate corrected FPD (cFPD) between control (n=26) and LQTS2-CM clusters (n=45) (B) and differences between asymptomatic (n=15) and symptomatic (n=25) LQTS2 CM clusters (C).

Figure 2: Schematic representation of detected novel SNPs that span exons and introns across KCNH2 gene. Numerical numbers denote intronic SNPs (1 , 2, 3, 4, 5, 6, 8) and coding region SNPs (7, 9, 10, 11 ) with details of SNPs depicted in Table 2. Definitions

As used herein, the term "comprising" or "including" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present revelation, the term "comprising" or "including" also includes "consisting of. The variations of the word "comprising", such as "comprise" and "comprises", and "including", such as "include" and "includes", have correspondingly varied meanings.

A family member includes biological grandparent(s), parent(s), aunts, uncles, siblings, cousin(s), child(ren) and grandchild(ren) Other biological relatives may also be considered as a family member, especially if there is a family history of LQTS2, cardiac arrhythmia and/or syncope.

A healthy subject without LQTS2 is a subject expressing:

SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3 and/or SEQ ID NO: 4. SEQ ID NO: 1 (potassium voltage-gated channel subfamily H member 2 isoform a fHomo sapiensl: NCBI Reference Seguence: NP 000229.1 ) PVRRGHVAPQNTFLDTIIRKFEGQSRKFIIANARVENCAVIYCNDGFCELCGYSRAEVMQRPCTCDFLH GPRTQRRAAAQIAQALLGAEERKVEIAFYRKDGSCFLCLVDWPVKNEDGAVIMFILNFEWMEKDMVGS PAHDTNHRGPPTSWLAPGRAKTFRLKLPALLALTARESSVRSGGAGGAGAPGAVWDVDLTPAAPSSESL ALDEVTAMDNHVAGLGPAEERRALVGPGSPPRSAPGQLPSPRAHSLNPDASGSSCSLARTRSRESCASVR RASSADDIEAMRAGVLPPPPRHASTGAMHPLRSGLLNSTSDSDLVRYRTISKIPQITLNFVDLKGDPFLA SPTSDREIIAPKIKERTH VTEKVTQVLSLGADVLPEYKLQAPRIHRWTILHYSPFKAV DWLILLLVIY TAVFTPYSAAFLLKETEEGPPATECGYACQPLAWDLIWIMFIVDILINFRTTYVNANEEWSHPGRIA VHYFKGWFLIDMVAAIPFDLLIFGSGSEELIGLLKTARLLRLVRVARKLDRYSEYGAAVLFLLMCTFALI AH LACIWYAIG MEQPH DSRIG LHNLGDQIGKPYNSSGLGGPSIKDKYVTALYFTFSSLTSVGFGNV SPNTNSEKIFSIC\MLIGSLMYASIFG VSAIIQRLYSGTARYHTQMLR\/REFIRFHQIPNPLRQRLEEY FQHAWSYTNGID AA/LKGFPECLQADICLHLNRSLLQHCKPFRGATKGCLRALAMKFKTTHAPPGDTLV HAGDLLTALYFISRGSIEILRGDVWAILGKNDIFGEPLNLYARPGKSNGDVRALTYCDLHKIHRDDLLE VLDMYPEFSDHF SSLEITFNLRDT MIPGSPGSTELEGGFSRQRKRKLSFRRRTDKDTEQPGEVSALGP GRAGAGPSSRGRPGGPWGESPSSGPSSPESSEDEGPGRSSSPLRLVPFSSPRPPGEPPGGEPLMEDCEKS SDTCNPLSGAFSGVSNIFSFWGDSRGRQYQELPRCPAPTPSLLNIPLSSPGRRPRGDVESRLDALQRQLN RLETRLSADMATVLQLLQRQMTLVPPAYSAVTTPGPGPTSTSPLLPVSPLPTLTLDSLSQVSQFMACEEL PPGAPELPQEGPTRRLSLPGQLGALTSQPLHRHGSDPGS

SEQ ID NO: 2 (potassium voltage-gated channel subfamily H member 2 isoform b [Homo sapiens!: NCBI Reference Seouence: NP 742053.1 )

MPVRRGHVAPQNTFLDTIIRKFEGQSRKFIIANARVENCAVIYCNDGFCELCGYSRAEVMQRPCTCDFLH GPRTQRRAAAQIAQALLGAEERKVEIAFYRKDGSCFLCLVDWPVKNEDGAVIMFILNFEWMEKDMVGS PAHDTNHRGPPTS LAPGRAKTFRL LPALLALTARESSVRSGGAGGAGAPGAVWDVDLTPAAPSSESL ALDEVTAMDNHVAGLGPAEERRALVGPGSPPRSAPGQLPSPRAHSLNPDASGSSCSLARTRSRESCAS RASSADDIEAMRAGVLPPPPRHASTGAMHPLRSGLLNSTSDSDLVRYRTISKIPQITLNFVDLKGDPFLA SPTSDREIIAPKIKERTHNVTEKVTQVLSLGADVLPEYKLQAPRIHRWTILHYSPFKAVWD LILLLVIY TAVFTPYSAAFLLKETEEGPPATECGYACQPLAVWLIVDIMFIVDILINFRTTYVNANEEWSHPGRIA WYFKGWFLIDMVAAIPFDLLIFGSGSEELIGLLKTARLLRLVRVARKLDRYSEYGAA^FLLMCTFALI AHWLACIWYAIG MEQPHMDSRIGWLHNLGDQIGKPYNSSGLGGPSIKDKYVTALYFTFSSLTSVGFGNV SPNTNSEKIFSICVMLIGSLMYASIFGNVSAIIQRLYSGTARYHTQ LRVREFIRFHQIPNPLRQRLEEY FQHAWSYTNGIDMNAVLKGFPECLQADICLHLNRSLLQHCKPFRGATKGCLRALAMKFKTTHAPPGDTLV HAGDLLTALYFISRGSIEILRGD\AAAILGMGWGAGTGLEMPSAASRGASLLNMQSLGLWTWDCLQGHWA PLIHLNSGPPSGAMERSPTWGEAAELWGSHILLPFRIRHKQTLFASLK SEQ ID NO: 3 (potassium voltage-gated channel subfamily H member 2 isoform c iHomo sapiensl; NCBI Reference Seguence: NP 742054.1 )

MAAPAGKASRTGALRPRAQKGRWRAVRISSLVAQEA/LSLGADVLPEYKLQAPRIHR TILHYSPFKAW DWLILLLVIYTAVFTPYSAAFLLKETEEGPPATECGYACQPLAWDLIA/DIMFIVDILINFRTTYVNA E EWSHPGRIAVHYFKGWFLIDMVAAIPFDLLIFGSGSEELIGLLKTARLLRLVRVARKLDRYSEYGAA\/L FLLMCTFALIAHWLACIWYAIGNMEQPHMDSRIGWLHNLGDQIGKPYNSSGLGGPSIKDKYVTALYFTFS SLTSVGFG VSPNTNSEKIFSICVMLIGSLMYASIFG VSAIIQRLYSGTARYHTQMLR EFIRFHQIP NPLRQRLEEYFQHAWSYTNGIDM AVLKGFPECLQADICLHLNRSLLQHCKPFRGATKGCLRALAMKFKT THAPPGDTLVHAGDLLTALYFISRGSIEILRGD\AAAILGKNDIFGEPLNLYARPGKSNGDVRALTYCDL HKIHRDDLLEVLDMYPEFSDHFWSSLEITFNLRDTNMIPGSPGSTELEGGFSRQRKRKLSFRRRTDKDTE QPGEVSALGPGRAGAGPSSRGRPGGPWGESPSSGPSSPESSEDEGPGRSSSPLRLVPFSSPRPPGEPPGG EPLMEDCEKSSDTCNPLSGAFSGVSNIFSFWGDSRGRQYQELPRCPAPTPSLLNIPLSSPGRRPRGDVES RLDALQRQLNRLETRLSADMAT\^LQLLQRQ LVPPAYSAVTTPGPGPTSTSPLLPVSPLPTLTLDSLSQ VSQF ACEELPPGAPELPQEGPTRRLSLPGQLGALTSQPLHRHGSDPGS

SEQ ID NO: 4 (potassium voltage-gated channel subfamily H member 2 isoform d fHomo sapiensl: NCBI Reference Sequence: NP 00119 727.1 )

MAAPAGKASRTGALRPRAQKGRWRAVRISSLVAQE\/LSLGADVLPEYKLQAPRIHRWTILHYSPFKAVW D LILLLVIYTAVFTPYSAAFLLKETEEGPPATECGYACQPLAVVDLIVDIMFIVDILINFRTTYV ANE EWSHPGRIAVHYFKGWFLIDMVAAIPFDLLIFGSGSEELIGLLKTARLLRLVRVARKLDRYSEYGAAVL FLLMCTFALIAHWLACIWYAIG MEQPHMDSRIGWLHNLGDQIGKPYNSSGLGGPSIKDKYVTALYFTFS SLTSVGFGWSPNTNSEKIFSICA/MLIGSLMYASIFGNVSAIIQRLYSGTARYHTQMLRVREFIRFHQIP NPLRQRLEEYFQHAWSYTNGIDM AVLKGFPECLQADICLHLNRSLLQHCKPFRGATKGCLRALAMKFKT THAPPGDTLVHAGDLLTALYFISRGSIEILRGDVWAILGMGWGAGTGLEMPSAASRGASLLNMQSLGL TWDCLQGHWAPLIHLNSGPPSGAMERSPTWGEAAELWGSHILLPFRIRHKQTLFASL

An asymptomatic LQTS2 (also known as low-risk LQTS2 subject) is a subject expressing a sequence having at least one amino acid differing from SEQ ID NO: 1 ; a sequence having at least one amino acid differing from SEQ ID NO: 2; a sequence having at least one amino acid differing from SEQ ID NO: 3 and/or a sequence having at least one amino acid differing from SEQ ID NO: 4 and not exhibiting at least one symptom selected from the group consisting of cardiac arrhythmia and/or syncope. Detailed description of the invention

According to a first aspect, the present invention provides a method for predicting and/or diagnosing long QT syndrome 2 (LQTS2) comprising identifying a subject having one or more SNP variants selected from the group: a heterozygous CT or a homozygous TT for rs3807375, a heterozygous CT or a homozygous TT for rs2968857, a heterozygous GA or a homozygous AA for rs34657537, a heterozygous AG or a homozygous GG for rs12668582, a heterozygous TC or a homozygous CC for rs4725385, a heterozygous GA or a homozygous AA for rs2269001 , a heterozygous GA or a homozygous AA for rs1805120, a heterozygous CT or a homozygous TT for rs188262457, a heterozygous TC or a homozygous CC for rs1805121 , a heterozygous AG or a homozygous GG for rs1137617; and a heterozygous GA, GC, AC or a homozygous CC or ΔΔ for rs531460655; wherein Δ comprises a deletion; s being at risk of or suffering from LQTS2. For the first aspect of the invention, the one or more SNP variants may comprise any number of the SNP variants in any combination. For example, the one or more variants may comprise any one or any two of the SNP variants.

For example, the one or more SNP variants may comprise a heterozygous CT or a homozygous TT for rs3807375; and/or a heterozygous CT or a homozygous TT for rs2968857.

In particular, one or more SNP variants may comprise a heterozygous CT or a homozygous TT for rs3807375. Alternatively, the one or more SNP variants may comprise a heterozygous CT or a homozygous TT for rs2968857. The one or more SNP variants may comprise a heterozygous CT or a homozygous TT for rs3807375 and a heterozygous CT or a homozygous TT for rs2968857.

According to a second aspect, the present invention provides a method for genotyping and thereby classifying a subject comprising:

Identifying (A) a subject having: a homozygous CC for SNP rs372582297; and a homozygous TT for rs11766022; or (B) a subject having (i) a heterozygous CG or a homozygous GG for rs372582297 and/or a heterozygous TG or a homozygous GG for rs372582297; and (ii) one or more variant SNPs selected from the group consisting of: a heterozygous CG or a homozygous GG for rs35277497, a heterozygous CT or a homozygous TT for rs1036145, a heterozygous GA or a homozygous AA for rs11763131 , a heterozygous AT or a homozygous TT for rs13244889; as a healthy subject without LQTS2 or an asymptomatic LQTS2 subject.

For the second aspect of the invention, the one or more variant SNPs in (ii) comprise any one SNP, any two SNPs, any three SNPs selected from or all four SNPs from the group.

Symptomatic LQTS2 subject typically shows symptoms such as cardiac arrhythmia and/or syncope. The method may be performed on a subject not exhibiting such symptoms. For a subject not exhibiting cardiac arrhythmia and/or syncope, a subject expressing at least one protein selecting from the group consisting of SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4 is a healthy subject.. On the other hand, a subject not exhibiting cardiac arrhythmia and/or syncope but expressing at least one protein with a sequence selected from the group consisting of a sequence having at least one amino acid differing from SEQ ID NO: 1 ; a sequence having at least one amino acid differing from SEQ ID NO: 2; a sequence having at least one amino acid differing from SEQ ID NO: 3 and/or a sequence having at least one amino acid differing from SEQ ID NO: 4 is an asymptomatic LQTS2. The method of the invention may be performed for a subject with at least one family member expressing at least one protein with a sequence selected from the group consisting of a sequence having at least one amino acid differing from SEQ ID NO: 1 ; a sequence having at least one amino acid differing from SEQ ID NO: 2; a sequence having at least one amino acid differing from SEQ ID NO: 3 and/or a sequence having at least one amino acid differing from SEQ ID NO: 4 and/or exhibiting cardiac arrhythimia and/or syncope.

The method of the invention may be applied in a population setting. The method may be conveniently applied to a number of subjects (a population). The method according to any aspect of the invention and/or protein expression where applicable is performed on an isolated sample from the subject.

Any suitable method may be applied to detect the SNP including but not limited to hybridization, primer extension, ligation, invasive cleavage, sequencing, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), multiplex ligation-dependent probe amplification (MLPA), micro PCR systems, microfluidic chip systems, allele specific amplification, snapshot mini- sequencing, strand displacement amplification, transcriptional mediated amplification, nucleic acid sequence-based amplification and/or helicase dependent amplification. It will be appreciated that each SNP for the first aspect of the invention may be detected by performing a PCR using the applicable primer pair in Table 1 (B) and then sequencing to determine the SNP.

It will also be appreciated that each SNP for the second aspect of the invention may be detected by performing a PCR using the applicable primer pair in Table 1(A) and then sequencing to determine the SNP.

The invention also includes a probe for detecting a SNP according to any aspect of the invention. The kit may comprise t probe(s) according to the invention. Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

Examples

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook (2012). Example 1 : Materials and methods

Cell culture

Fibroblast were cultured in 10% FBS containing DMEM medium with high glucose supplemented with 1 % non-essential amino acids, 10mM L-Glutamine and 1X Penicillin-Streptomycin. Fibroblast were seeded at a density of 5000 cells/cm² and cultured for 5-7 days. Cells were trypsinized and collect at 80% confluency and these cells were washed with PBS and used for subsequent studies.

DNA extraction DNA extraction was performed using DNAeasy extraction kit (Qiagen) as per the manufacturer's instruction. The quality and quantity of DNA was evaluated by Nanodrop (Thermo fisher).

Next generation sequencing

Fibroblast from patient samples were collected and genomic DNA extraction was performed using with Epicentre® Nextera™ DNA library kit (lllumina) for sequencing on an lllumina® system as per the manufacturer's recommendation. Sequencing was performed for KCNH2 gene with specific designed primers. The data set were then analyzed with reference alignment to the human reference ChGr38/hg19 using the Burroughs-Wheeler Aligner, SAMtools. Using maximum likelihood estimation model using a custom Java 6.0 tool that treated each SNP as an independent measure of probability for the characteristic evaluated and compared with the existing database or previously published data for SNP frequencies and correlation to phenotype. The novel SNPs are compared to published databases of KCNH2 from Ensembl project. PCR studies and DNA sequencing Genomic DNA was extracted from patient and normal fibroblasts using DNeasy genomic extraction kits as per the manufacturer's instruction. Post-extraction DNA was quantitated and subjected to SNP specific PCR amplification using standard PCR reagents and primers (table 1 ). Samples were cycled as follows: 5 min at 95°C, followed by 40 cycles of 20 s at 95°C, 30 s at 60°C, 30 s at 72°C, followed by a final extension cycle of 7min at 72°C. PCR amplicons were purified and send for DNA sequencing from commercial sources.

Table 1 : List of SNP primers used in the study (A)

SNP Forward primer Reverse Primer

number

SNP 1.1 ACCTTCCTGGACACCATCATCCG GGAATTAGGTGACAGTCTCCAGGGC

(SEQ ID NO: 5)

(SEQ ID NO: 6)

SNP 1.2 CGGGCTGATCTTGTATCTTGCAACC AAGGTGCACACAGCTGCCACTGTC

(SEQ ID NO: 7) (SEQ ID NO: 8)

SNP 2.1 ACCTTCCTGGACACCATCATCCG GGAATTAGGTGACAGTCTCCAGGGC

(SEQ ID NO: 9) (SEQ ID NO: 10)

SNP 2.2 TGGGACTTCCAGTTCGTGCACC GAAAGGCCAGGGACATTGCAGG

(SEQ ID NO: 12)

(SEQ ID NO: 11 )

SNP 5.2 ACTGCAACAGCTGATGCTGCGG ATCGCTCTGGTCTGCCTCCTATCTC

(SEQ ID NO: 13) (SEQ ID NO: 14)

SNP 6.2 CCGCATATCTTGGCCACTCTCG CCCTACTTG AC ACCAC ATACGG G G

(SEQ ID NO: 15) (SEQ ID NO: 16) (B)

SNP GrCh38 Forward primer Reverse Primer

number Position

1 150970122 AGTTTGATGGTGGCCTTGGGCTCTG ATGGAGGGGACTTTAGGGGAGCACC

(SEQ ID O: 17) (SEQ ID NO: 18)

2 150965242 ACCTTCCCAGGCTTCTCAGCAGGAG ACAGGGAGAGAGTGTGTGTGTGCGC

(SEQ ID NO: 19) (SEQ ID NO: 20)

3 150960720 AACTGAGTGCTTGGCCTAAAAGGGG CCACTCAGTGGGCAGAGCAGAAAGC

(SEQ ID NO: 21 ) (SEQ ID NO: 22)

4 150960113 AACTGAGTGCTTGGCCTAAAAGGGG CCACTCAGTGGGCAGAGCAGAAAGC

(SEQ ID NO: 23) (SEQ ID NO: 24)

5 150957732 GGTGCCTGGGAGATGGAAATCC TCCGAGGTGGAGTTGAGCAAGC

(SEQ ID NO: 25) (SEQ ID NO:26)

6 150954728 CTGGAATGTGCGGAAGCCACTGTGT CTCTG C AG CCACC AG CTC ACCTATG

(SEQ ID NO: 27) (SEQ ID NO: 28)

7 150952443 CTGCTGAAGGAGACGGAAGAAGGCC AGTTGGTGTTGGGAGAGACGTTGCC

(SEQ ID NO: 29) (SEQ ID NO: 30)

8 150952318 CTGCTGAAGGAGACGGAAGAAGGCC AGTTGGTGTTGGGAGAGACGTTGCC

(SEQ ID NO: 31 ) (SEQ ID NO: 32)

9 150951701 CTGCTGAAGGAGACGGAAGAAGGCC AGTTGGTGTTGGGAGAGACGTTGCC

(SEQ ID NO: 33) (SEQ ID NO:34)

10 1509511 10 TAGAGACTCCAGGGGCCACCATCGT GTTTGCTGTGCCAAGAGGTTCCCCT

(SEQ ID NO: 35) (SEQ ID NO: 36)

1 1 150945480 TCTACTTCCCACCTTGGTGCCTCCC AGTTCCTCTCCCCTTCCACGGTCAG

(SEQ ID NO: 37) (SEQ ID NO:38) Human iPSC generation and cardiomyocyte differentiation

Human skin cells were reprogrammed to human induced pluripotent stem cell (hiPSCs) as described previously (Mehta et al., 2014). These hiPSC were differentiated into cardiomyocytes as described previously (Mehta et al., 2014a). Multielectrode array (MEA)

Electrophysiological evaluation of cardiomyocytes were performed using MEA system as reported previously (Mehta et al., 2013). The corrected field potential duration (cFPD) which is similar to QTc in clinical set up were used to identify the QT prolongation in LQTS2 patient derived cardiomyocytes (Mehta et al., 2014a).

Example 2: Results Initial study

In the initial study, fibroblasts from 1 normal healthy individual (no genetic mutation) and 5 clinically diagnosed LQTS2 patients (confirmed by genetic mutation) were collected. Targeted whole genome next generation sequencing for KCNH2 gene was performed. Table 2 provides the list of samples utilized in the initial study with their mutation as well as clinical manifestations. Table 2: Clinical information on patient samples

In a clinical setup, LQTS2 patients are generally identified based on long QTc interval in their ECG followed by a genetic screening to identify the gene mutation. However, prolonged QTc interval does not discriminate between asymptomatic and symptomatic cases, which renders disease management challenging. Viral free hiPSC were also generated from the fibroblasts from the 5 LQTS2 patients (Table 2) and used to derive cardiomyocytes. LQTS2 patient specific hiPSC derived cardiomyocytes from 5 patients (Table 2) were evaluated to determine if symptomatic and asymptomatic cases could be discriminated based on eietrophysiological evaluations. Patients 2 and 3 are symptomatic LQST2 patients; while patients 4-6 were asymptomatic, MEA studies demonstrate that while there was a stark difference between the cFPD (similar to QTc) in controls and LQTS2 (Figure 1 ), no significant change was noted between asymptomatic and symptomatic cases (Figure 1 ). These findings are similar to the clinical setup, demonstrating, other factors such as SNP may be contributing to the disease penetrance.

SNP data analysis from NGS from a total of six samples indicates marked differences in the SNP profile of control healthy individual (Fib-1 ) and LQTS2 patients (Fib-2 to -6). Expectedly, DNA sequence alignment demonstrates that normal healthy fibroblast share some common SNPs with LQTS2 samples (Figure 2). However, LQTS2 samples collected from different individuals having different mutations (Table 2) show similar SNPs that are not present in the control (Figure 2 & Table 3). A total of 1 1 novel SNPs were detected to be associated with LQTS2, as indicated in rectangular bars in Figure 2. While 7 SNPs were found in the intronic region, 4 of them were in the coding regions of KCNH2 gene. Out of the total of 7 LQTS2 specific intronic SNPs, 4 SNPs were found in the intron 2 of the gene. Most of these SNPs demonstrated a similar change across all samples for replacing a C to T or a G to A (Table 3), except the SNP found in exon 15 (C to G). These results suggest that most of these SNPs may have probable regulatory role by modifying the CpG signature and these SNPs may modulate the methylation status of the KCNH2 gene thereby influencing its transcriptional activity, which may play a role in regulating LQTS2 in patients. Table 3 outlines the position and SNP change information for these 11 SNPs based on NCBI database http://www.ncbi. nlm.nih.gov/gene/?term=3757 (KCNH2 Gene ID: 3757). These data also demonstrate the presence of SNPs in the exons of KCNH2 gene, however, they are found to be synonymous SNPs that do not result in change of the amino acid sequence of the protein and are deemed not responsible for the mutation occurred in these LQTS2 samples. Table 3: Position and details of detected novel SNPs in KCNH2

Protective and Predictive SNPs

In the next phase, asymptomatic LQTS2 and symptomatic LQTS2 patients were segregated and re-evaluated NGS SNP data for SNPs specific for each group. Importantly, several unique SNPs were identified to segregate asymptomatic patients from symptomatic LQTS2 patients whereby some SNPs could distinctly predict the asymptomatic LQTS2 patients (Table 4) from the symptomatic LQTS2 patients (Table 5). Table 4 shows a list of SNPs which appears to convey a protective function in asymptomatic patients and were denoted as protective SNPs, whereas SNPs in Table 5, were more pronounced in symptomatic cases and were labeled as Predictive SNPs (Table 5). Moreover, two (SNP 3.2 and 4.2 of Table 4) of the six protective SNPs demonstrated redundancy in detection as they did not provide addition information, thus, was eliminated from further studies. The results also suggested that the interplay of these protective and predictive SNPs could be utilized for the early diagnosis of patients suffering from LQTS2 as well as help in risk stratification of these patients.

Table 4: Position of detected novel protective SNPs in LQTS2 patients

In this initial study, a cohort of LQTS2 patients with varying disease expressivity was investigaed, it was posited that specific SNPs in KCNH2 gene may have probable roles in increased individual's susceptibility to arrhythmias. These SNPs, when validated, could prove a valuable prognosticating tool to predict likelihood of arrhythmic episodes in patients with genetically inherited LQTS2 and patients that are prone to symptoms of acquired LQTS through QT- prolonging drugs or metabolic derangements such as hyperkalemia or dysfunctional electrolyte imbalance.

In order to validate this, a total of 46 patient DNA samples were collected. Out of these 46 patient samples, 14 samples were controls as they were not known to have any KCNH2 mutation, whereas 32 samples showed presence of known KCNH2 mutations confirming LQTS2 phenotype (Table 6). All these samples were subjected to SNP identification by DNA sequencing. SNP identification of the two predictive SNPs was first performed followed by protective SNPs on all 46 samples. If the sample from a subject did not contain any predictive SNPs, the subject was classified as a healthy subject without LQTS2 or an asymptomatic LQTS2 subject. The results demonstrated that all 14 control samples were negative for predictive SNPs, as expected, demonstrating that all 14 samples would have been correctly classified (i.e. with no disease manifestation). Some of the control patients expressed protective SNPs (Table 6).

Among the 32 LQTS2 samples, there was a mixed set of genetically identified and clinically classified asymptomatic (16 patients) and symptomatic (16 patients). In these LQTS2 groups, patients that did not show predictive SNPs like patient sample H49, PJ3 and PJ4, were classified as asymptomatic patients outright. However, patients that showed the presence of predictive SNPs like patient sample H26 and PJ10 (Table 6) were stratified further by relying on the presence of protective SNPs. It is well known clinically that the LQTS2 disease severity spectrum is very broad, it is possible that these protective SNPs may counterbalance the presence of the predictive SNPs that render the disease asymptomatic. Therefore, patient H26 and PJ10 was predicted to be asymptomatic due to the co-segregation of predictive and protective SNP signature. Based on this understanding of unique SNP signature, 16 symptomatic LQTS2 patients were considered. Only 5 of the 16 symptomatic patients followed this prediction, as they lacked protective SNPs but expressed predictive SNPs (patient samples H36, H48 and PJ7). However, the other symptomatic patients could not be correctly identified based on the combined SNP (predictive and protective) signature, suggesting that there may be other genes or modifiers that may be contributing to the disease manifestation in symptomatic LQTS2 patients. Our SNP prediction system was able to identify all 16 asymptomatic patients correctly with 100% sensitivity (Table 6).

Table 6: SNP analysis of 46 patient samples

Samples showing ++ are homozygous for SNP whereas +- denotes heterozygous for SNP and - denotes absence of SNPs. ND is not detected. The table also shows comparison between our prediction and clinical classification of the patients. AS: asymptomatic; S: symptomatic Discussion

It has previously been demonstrated that a common KCNH2 K897T polymorphism modified a latent LQTS2 mutation and a KCNE1 D85N polymorphism aggravated KCNQ1 mutations by significantly increasing the risk of arrhythmia (Crotti et a/., 2005) while rs2074238 polymorphism of KCNQ1 was found to confer protective effect against LQTS event (Duchatelet et a/., 2013). In fact, a SNP variant within exon 6 of KCNH2 (Wang et al 2009) and another 2 SNPs in high linkage disequilibrium with KCNH2 (Andreasen et al., 2013) have been found to predispose patient to atrial arrhythmia. Collectively, these data strongly support the important of genetic modifiers in disease penetrance and expressivity in LQTS.

The newly identified novel SNP in the intron 2 (SNP#2 Table 3) is predicted to reside within a putative NKx2.5 cardiac transcription factor-binding region, which suggests a tightly regulated KCNH2 expression in cardiomyocytes. This couples with another intron 2 SNP (SNP#3 Table 3) that resides within a CpG island that is predicted to shift methylation status and gene expressivity of KCHN2 that could together affect hERG currents that determines cardiac repolarization event leading up to presentation of LQTS2 symptoms. Consistently, it was previously demonstrated that heterozygous overexpression of A561V mutant KCNH2 allele not only affected hERG currents to result in prolonged repolarization, but also impacted outcome of arrhythmogenesis, in LQTS2 patient-derived cardiomyocytes (Mehta et al 2014).

It is envisaged that selective interrogation of such SNPs of KCHN2 gene would be cost-effective as part of a protocol for pre-emptive prognosis of malignant arrhythmia to risk stratify patients at risk of manifesting LQTS2 symptoms. The segregation of SNPs that demarcates asymptomatic and symptomatic LQTS2 patients is crucial in implementing such stratification strategy. The unique SNPs found in this study that identify asymptomatic LQTS2 from symptomatic LQTS2 (Tables 4 and 5) could contribute significantly towards personalized medicine in individuals most at risk of arrhythmia.

References

Any listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that such document is part of the state of the art or is common general knowledge.

Andreasen et al., (2013) Genetic modifier of the QTc interval associated with early-onset atrial fibrillation; Canadian Journal of Cardiology 29:1234-40. Crotti et al., (2005) KCNH2-K897T is a genetic modifier of latent congenital long-QT syndrome; Circulation 1 12:1251-58.

Duchatelet et al., (2013) Identification of a KCNQ1 polymorphism acting as a protective modifier against arrhythmic risk in long QT syndrome; Circ Cardiovasc Genet 6:354-61 . Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012). Mehta et al., (2013) Pharmacol-Electrophysiology of Viral-Free Induced Pluripotent Stem Cell-derived Human Cardiomyocytes; Toxicol Sci 131 :458-69.

Mehta et al., (2014a) Phasic Modulation of Wnt Signaling Enhances Cardiac Differentiation in Human Pluripotent Stem Cells by Recapitulating Developmental Ontogeny; BBA-Molecular Cell Research 1843:2394-2402.

Mehta et al. (2014b) Re-trafficking of hERG reverses long QT syndrome 2 phenotype in human iPS-derived cardiomyocytes; Cardiovasc Res 102:497-506.

Schwartz and Crotti (2011 ) QTC behavior during exercise and genetic testing for the long-QT syndrome; Circulartion 124:2181 -2184. Vincent (2002) The long QT syndrome; Indian Pacing Electrophysiol J. 2(4):127-142.

Wang et al., (2009) Genetic polymorphism of KCNH2 confers predisposition of acquired atrial fibrillation in Chinese; J Cardiovasc Electrophysiol 20:1 158-62.

Claims

1. A method for predicting and/or diagnosing long QT syndrome 2 (LQTS2) comprising identifying a subject having one or more SNP variants selected from the group: a heterozygous CT or a homozygous TT for rs3807375, a heterozygous CT or a homozygous TT for rs2968857, a heterozygous GA or a homozygous AA for rs34657537, a heterozygous AG or a homozygous GG for rs12668582, a heterozygous TC or a homozygous CC for rs4725385, a heterozygous GA or a homozygous AA for rs2269001 , a heterozygous GA or a homozygous AA for rs1805120, a heterozygous CT or a homozygous TT for rs188262457, a heterozygous TC or a homozygous CC for rs1805121 , a heterozygous AG or a homozygous GG for rs1137617; and a heterozygous GA, GC, AC or a homozygous CC or ΔΔ for rs531460655; wherein Δ comprises a deletion; as being at risk of or suffering from LQTS2.

2. The method according to claim 1 , wherein the one or more SNP variants comprise a heterozygous CT or a homozygous TT for rs3807375; and/or a heterozygous CT or a homozygous TT for rs2968857.

3. The method according to claim 1 or 2, wherein the one or more SNP variants comprise a heterozygous CT or a homozygous TT for rs3807375.

4. The method according to claim 1 or 2, wherein wherein the one or more SNP comprise a heterozygous CT or a homozygous TT for rs2968857.

5. The method according to 1 or 2, wherein the one or more SNP variants comprise a heterozygous CT or a homozygous TT for rs3807375 and a heterozygous CT or a homozygous TT for rs2968857

6. A method for genotyping and thereby classifying a subject comprising:

Identifying (A) a subject having: a homozygous CC for rs372582297; and

a homozygous TT for rs11766022; or (B) a subject having (i) a heterozygous CG or a homozygous GG for rs372582297 and/or a heterozygous TG or a homozygous GG for rs372582297; and (ii) one or more variant SNPs selected from the group consisting of: a heterozygous CG or a homozygous GG for rs35277497, a heterozygous CT or a homozygous TT for rs1036145, a heterozygous GA or a homozygous AA for rs11763131 , a heterozygous AT or a homozygous TT for rs13244889; as a healthy subject without LQTS2 or an asymptomatic LQTS2 subject.

7. The method according to claim 6, wherein the one or more variant SNPs in (ii) comprise only one SNP selected from the group.

8. The method according to claim 6, wherein the one or more variant SNPs in (ii) comprise any two SNPs selected from the group.

9. The method according to claim 6, wherein the one or more variant SNPs in (ii) comprise any three SNPs selected from the group.

10. The method according to claim 6, wherein the one or more variant SNPs in (ii) comprise all four SNPs from the group.

11. The method according to any one of claims 6 to 10, wherein the method is performed on a subject not exhibiting at least one symptom selected from the group consisting of cardiac arrhythmia and syncope; wherein a subject expressing at least one protein selecting from the group consisting of SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4 is a healthy subject; or wherein a subject expressing at least one protein with a sequence selected from the group consisting of a sequence having at least one amino acid differing from SEQ ID NO: 1 ; a sequence having at least one amino acid differing from SEQ ID NO: 2; a sequence having at least one amino acid differing from SEQ ID NO: 3 and/or a sequence having at least one amino acid differing from SEQ ID NO: 4 is an asymptomatic LQTS2.

12. The method according to any one of claims 1 to 11 , wherein said method is performed on an isolated sample from the subject.

13. The method according to any one of claims 1 to 11 , wherein said method is performed for a subject with at least one family member expressing at least one protein with a sequence selected from the group consisting of a sequence having at least one amino acid differing from SEQ ID NO: 1 ; a sequence having at least one amino acid differing from SEQ ID NO: 2; a sequence having at least one amino acid differing from SEQ ID NO: 3 and/or a sequence having at least one amino acid differing from SEQ ID NO: 4 and/or exhibiting cardiac arrhythimia and/or syncope