WO2012149406A2 - Assessing and treating humans with long qt syndrome - Google Patents

Abstract

This document provides methods and materials involved in assessing and treating humans with LQTS or with a potential mutation in a KCNQ1 nucleic acid that encodes a Kv7.1 potassium channel subunit. For example, methods and materials for determining if a human containing a mutation in a KCNQ1 nucleic acid that encodes a K_v7.1 potassium channel subunit on one allele also contains, on the same allele (a cis relationship) or on the other allele (a trans relationship), a genetic variation (e.g., a SNP) in a 3' UTR of KCNQ1 nucleic acid that creates a miR-378 binding site are provided.

Description

ASSESSING AND TREATING HUMANS WITH LONG QT SYNDROME

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 61/480,962, filed April 29, 201 1. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in assessing and treating humans with long QT syndrome (LQTS) or with a potential mutation in a KCNQ 1 nucleic acid that encodes a K_v7.1 potassium channel subunit. For example, this document provides methods and materials for determining if a human containing a mutation in a KCNQ1 nucleic acid that encodes a K_v7.1 potassium channel subunit on one allele also contains, on the same allele (a cis relationship) or on the other allele (a trans relationship), a genetic variation (e.g., a SNP) in a 3 ' UTR of KCNQ 1 nucleic acid that creates a new microRNA (e.g., miR-378) binding site.

2. Background Information

LQTS is a common heritable cardiac channelopathy that can cause premature sudden death due to life-threatening arrhythmias in relation to prolongation of the heart rate-corrected QT interval (QTc). The most prevalent form, LQT1, is caused by loss-of- function mutations in the KCNQ 1 -encoded K_v7.1 potassium channel (IKs). LQT1 is characterized by incomplete penetrance and variable expressivity whereby family members carrying identical mutations have profound differences in their QTc and clinical course. The cause for this heterogeneity remains largely elusive. K_v7.1 is a tetrameric channel derived from the post-translational assembly of four KCNQ 1 -encoded subunits. Therefore, patients heterozygous for an LQT1 -causative mutation combine the translated products from normal and LQT1 -mutation-containing alleles to form tetrameric channels. If both alleles are similarly expressed, one would predict that 1/16th of the K_v7.1 channels stem solely from the normal allele, and 1/16th of the K_v7.1 channels stem solely from the mutated allele. The remaining channels would be hybrids of the mutated and healthy alleles.

SUMMARY

This document relates to methods and materials involved in assessing and treating humans with LQTS or with a potential mutation in a KCNQ 1 nucleic acid that encodes a K_v7.1 potassium channel subunit. For example, this document provides methods and materials for determining if a human containing a mutation in a KCNQ 1 nucleic acid that encodes a K_v7.1 potassium channel subunit on one allele also contains, on the same allele (a cis relationship) or on the other allele (a trans relationship), a genetic variation (e.g., a SNP) in the 3' UTR of KCNQl nucleic acid that creates a new microRNA (e.g., a miR- 378) binding site.

For the purposes of this document, the term "KCNQ 1 mutation" refers to a genetic variation (e.g., a substitution, deletion, or insertion of a nucleotide or nucleotides) that is present within a KCNQl nucleic acid that encodes a K_v7.1 potassium channel subunit, causes an alteration in the amino acid sequence of a K_v7.1 potassium channel subunit, and is associated with LQTS. The term "KCNQl UTR variation" refers to a genetic variation (e.g., a substitution, deletion, or insertion of a nucleotide or nucleotides) that is present within the 3' untranslated region of a KCNQl nucleic acid and creates a microRNA binding site (e.g., a miR-378 binding site). As described herein, humans with a KCNQ l mutation that is present on the same allele that contains a KCNQ 1 UTR variation can experience less severe symptoms of LQTS if the other allele lacks KCNQl mutations (e.g., is a wild-type KCNQl nucleic acid). Humans with KCNQl nucleic acid lacking KCNQl mutations (e.g., a wild-type KCNQl nucleic acid that encodes a K_v7.1 potassium channel subunit) that is present on the same allele that contains a KCNQl UTR variation can experience more severe symptoms of LQTS if the other allele contains a KCNQl mutation. Thus, the cis or trans relationship of KCNQ 1 mutations and KCNQ 1 UTR variations can be used to assess the severity of LQTS.

Having the ability to determine the cis or trans relationship of KCNQ 1 mutations and KCNQl UTR variations within a human heterozygous for a KCNQl mutation and heterozygous for a KCNQ 1 UTR variation can allow clinicians and patients to determine appropriate levels of LQTS disease monitoring and care. For example, a patient identified as having increased LQTS severity as described herein can be selected for treatment with agents designed to inhibit microRNA activity.

In general, this document features a method for assessing the cis or trans nature of a human heterozygous for a KCNQ 1 mutation and heterozygous for a KCNQ 1 UTR variation. The method comprises, or consists essentially of, (a) determining if an allele of the human comprises (i) the KCNQ 1 mutation and the KCNQ 1 UTR variation, (ii) a lack of the KCNQl mutation and a lack of the KCNQl UTR variation, (iii) the KCNQl mutation and a lack of the KCNQl UTR variation, or (iv) the KCNQl UTR mutation and a lack of the KCNQl variation, (b) classifying the human as having the KCNQl mutation and the KCNQl UTR variation in cis if the allele comprises (i) or (ii), and (c) classifying the human as having the KCNQl mutation and the KCNQl UTR variation in trans if the allele comprises (iii) or (iv). The human can be an LQT1 patient. The allele can comprise the KCNQ 1 mutation and the KCNQ 1 UTR variation, and the human can be classified as having the KCNQ l mutation and the KCNQl UTR variation in cis. The allele can comprise a lack of the KCNQl mutation and a lack of the KCNQl UTR variation, and the human can be classified as having the KCNQl mutation and the KCNQl UTR variation in cis. The allele can comprise the KCNQl mutation and a lack of the KCNQl UTR variation, and the human can be classified as having the KCNQl mutation and the KCNQl UTR variation in trans. The allele can comprise the KCNQl UTR variation and a lack of the KCNQ 1 mutation, and the human can be classified as having the KCNQl mutation and the KCNQl UTR variation in trans. The KCNQl mutation can be L266P, R518X, G168R, or R594Q. The KCNQl UTR variation can be an rs2519184 SNP or rs8234 SNP.

In another aspect, this document features a method for treating a mammal having a KCNQl UTR variation that is part of a microRNA binding site, wherein the binding of a microRNA to the binding site reduces expression of a K_v7.1 potassium channel subunit lacking a mutation associated with LQTS. The method comprises, or consists essentially of, administering a miRNA inhibitor to the mammal under conditions wherein the miRNA inhibitor reduces the ability of the microRNA to reduce expression of the subunit. In another aspect, this document features a method for treating a human comprising, or consisting essentially of, administering an agent that inhibits or mimics the activity of a microR A that targets a KCNQ1 UTR variation to increase the ratio of expression of a non-mutated KCNQ 1 allele to mutated KCNQ 1 allele.

In another aspect, this document features a method for treating a human having long QT syndrome. The method comprises, or consists essentially of, (a) determining if an allele of the human comprises (i) the KCNQ1 mutation and the KCNQ1 UTR variation, (ii) a lack of the KCNQ 1 mutation and a lack of the KCNQ 1 UTR variation, (iii) the KCNQ1 mutation and a lack of the KCNQ1 UTR variation, or (iv) the KCNQ1 UTR mutation and a lack of the KCNQ 1 variation, and (b) administering a beta blocker to the human, implanting an implantable cardioverter-defibrillator into the human, or performing a sympathetic denervation procedure on the human. The method can comprise administering the beta blocker to the human. The beta blocker can be metoprolol, carvedilol, bisoprolol, nebivolol, or propanolol. The method can comprise implanting the implantable cardioverter-defibrillator into the human. The method can comprise performing the sympathetic denervation procedure on the human.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1. Genetic variation in the 3 'untranslated region oiKCNQl. (A) Single nucleotide polymorphisms (SNPs) found in the study cohorts of the Academic Medical Center Amsterdam (AMC) and the Mayo Clinic (MC). Position of the nucleotide change is starting from the ATG start codon ( CBI build 36, hgl 8). (B) Predicted binding between miR-378 and the 3'UTR oiKCNQl containing the minor A variant of SNP rs2519184 and the minor G variant of SNP rs8234. 'rs' numbers denote SNP identities from public database. Longer lightly shaded boxes represent exons, and darker shaded bars represent SNPs. (C) Relative expression of miR-378 in human donor hearts in comparison with other abundantly expressed cardiac miRNAs (miR-133b, miR-21, and miR-208b). Data are presented as mean ± standard error.

Figure 2. The functional effects of genetic variation in the 3 'untranslated region (3 'UTR) oiKCNQl. (A) Luciferase assays in neonatal rat heart-derived H10 cells transfected with two independent reporter plasmids containing either the major or the minor haplotype of SNPs in KCNQFs 3 'UTR. Observed differences in luciferase activity between the major and minor haplotype may indicate that translation is inhibited by enhanced miRNA binding to the 3'UTR. (B) Luciferase assays in H10 cells transfected with a reporter plasmid containing either the major haplotype of the 3 'UTR oiKCNQl or the same reporter where only one of the two SNPs predicted to affect miR-378 binding (rs2519184 or rs8234) was changed. Introducing either one of the minor SNP alleles was sufficient to decrease luciferase activity. (C) Luciferase assays in COS-7 cells cotransfected with the 3'UTR KCNQ1 reporter, containing either the major or the minor haplotype of SNPs in the 3'UTR oiKCNQl, and an expression vector for miR-378 or a control miRNA. Overexpression of miR-378 (but not the control miRNA) decreased luciferase activity of only the 3 'UTR KCNQ 1 reporter with the minor SNP haplotype. Data are presented as mean ± standard error. Darker shaded bar represents a major SNP variant, while the lighter shaded bar represents a minor SNP variant.

Figure 3. Allele-specific effects of SNPs rs2519184 and rs8234 on QTc. Allele- specific effects of SNP rs2519184 (A), SNP rs8234 (B), and haplotype of the two SNPs

(C) on QTc duration for the AMC population. Allele-specific effects of SNP rs2519184

(D) , SNP rs8234 (E), and haplotype of the two SNPs (F) on QTc duration for the MC population. The dashed line represents mean QTc of all individuals within the study population regardless of the specific LQT1 -causative mutation and the 3 'UTR SNP status. Numbers below genotypes denote group sizes. Data are presented as mean ± standard error. N, normal KCNQ1 allele; M, mutant KCNQ1 allele. Darker shaded box represents a major SNP variant, while the lighter shaded box represents a minor SNP variant.

Figure 4. Allele-specific effects of SNPs rs2519184 and rs8234 on QTc in two single families. Pedigree structure and allele-specific haplotype analysis of SNPs rs2519184 and rs8234 with regard to QTc are displayed for the largest family from AMC (A) and MC (B). Numbers above genotypes denote QTc values, 'n.a.' means not available. N, normal KCNQ1 allele; M, mutant KCNQ1 allele. Darker shaded box represents a major SNP variant, while the lighter shaded box represents a minor SNP variant.

Figure 5. Allele-specific effects of SNPs rs2519184 and rs8234 on

symptomatology. Allele-specific effects of SNPs rs2519184 and rs8234 with regard to the occurrence of cardiac symptoms (unexplained syncope, documented torsades de pointes ventricular tachycardia/ventricular fibrillation, and/or [aborted] sudden death) are shown in the total combined study population (AMC and MC). (A) Effects of SNP rs2519184. (B) Effects of SNP rs8234. (C) Allele-specific haplotype analysis of the two SNPs with regard to the occurrence of symptoms. Numbers below genotypes denote group sizes. Data are presented as mean ± standard error. N, normal KCNQ1 allele; M, mutant KCNQ1 allele. Darker shaded box represents a major SNP variant, while the lighter shaded box represents a minor SNP variant.

Figure 6. The functional effects of genetic variation in the 3 'untranslated region

(3 'UTR) oiKCNQl. (A) Luciferase assays in neonatal rat cardiomyocytes transfected with two independent reporter plasmids containing either the major or the minor haplotype of SNPs in the 3'UTR oiKCNQl. Observed differences in luciferase activity between the major and minor haplotype may indicate that translation is inhibited by enhanced miRNA binding to the 3 'UTR. (B) Luciferase assays in neonatal rat cardiomyocytes transfected with a reporter plasmid containing either the major haplotype of the 3'UTR oiKCNQl or the same reporter where only one of the two SNPs predicted to affect miR-378 binding (rs2519184 or rs8234) was changed. Introducing either one of the minor SNP alleles was sufficient to decrease luciferase activity. Data are presented as mean ± standard error. Darker shaded bars represent a major SNP variant, while the lighter shaded bars represent a minor SNP variant. Figure 7 is a diagram of a mechanism where SNPs in the 3 'UTR oiKCNQl modulate the assembly of the K_v7.1 potassium channel in type 1 long QT syndrome. Individuals with type long QT syndrome (LQT1) are heterozygous for the disease- causing mutation in KCNQ1. Four KCNQ1 -encoded subunits co-assemble post- trans lationally to form one tetrameric channel. Since the 3 'UTR plays an important role in messenger-RNA translation, SNPs in this region may influence repolarization by altering the balance and composition of the K_v7.1 tetramers derived from translation of the normal allele and the mutant allele. If SNPs exert no effect on messenger-RNA translation, the balance between normal and mutant subunits within each channel is equal. However, if the minor genotypes of the SNPs suppress messenger-RNA translation (by creating target sites for microRNAs as predicted for SNPs rs2519184 and rs8234; see Figure 2), then the balance between normal and mutant subunits within each channel depends on whether the 'suppressive SNPs' reside on the normal allele or the mutant allele. If the 'suppressive SNPs' reside on the normal allele, the number of normal subunits in the channels would decrease. Inversely, if the 'suppressive SNPs' reside on the mutant allele, this would decrease the relative number of mutant subunits and shift the K_v7.1 tetramers to a greater percent of normal allele-derived monomeric subunits.

Figure 8 is a graph showing inhibition by a microRNA inhibitor (e.g., an anti- miR) designed to inhibit miR-378, at two different dosages.

Figure 9 is a graph showing that the inhibition shown in Figure 8 prevents the suppressive effects of UTR variants. The two bars on the right (antimiR54) represent a control demonstrating that the effect (i.e., lack of suppression) only occurs after using the specific antimir against 378.

Figure 10 is a graph of luciferase assay results of cardiac myocytes with knockdown of miR-378.

Figure 1 1 is a graph demonstrating an allelic imbalance on mRNA levels.

DETAILED DESCRIPTION

This document relates to methods and materials involved in assessing and treating humans with LQTS or with a potential mutation in a KCNQ1 nucleic acid that encodes a K_v7.1 potassium channel subunit. For example, this document provides methods and materials for assessing LQTS severity by determining if a human containing a KCNQ1 mutation on one allele also contains, on the same allele (a cis relationship) or on the other allele (a trans relationship), a KCNQ 1 UTR variation. Figure 7 provides several examples of how the cis and trans relationship of KCNQ 1 mutations and KCNQ 1 UTR variations can influence LQTS severity.

Examples of KCNQ 1 mutations include, without limitation, those listed in Table A or described elsewhere (Kapplinger et ah, Heart Rhythm., 6(9): 1297-303 (2009)). Examples of KCNQ 1 UTR variations include, without limitation, the rs2519184 SNP, rs8234 SNP, and rs 10798 SNP. A KCNQ1 UTR variation can be any genetic variant present within the 3' untranslated region of a KCNQ1 nucleic acid that creates a microRNA binding site for a microRNA that is capable of reducing expression of mRNA to which it binds. Examples of such microRNA include, without limitation, miR-378, miR-328, and miR-220. Table A. KCNQ1 LQT1 -associated mutations.

Exon 1 273 299delCTCC V91fs+136X* Frame shift N-Terminal 1 ATCTACAGCAC GCGCC

GCCCGGTinsGG

Exon 1 332 A>G Y111C Missense N-Terminal 5

Exon 1 350 C>T P117L Missense N-Terminal 1

Exon 1 365insT 121fs+162X* Frame shift N-Terminal 2

Exon 1 381 C>A F127L* Missense SI Domain 1

Intron 386+1 G>A Splice site SI Domain 1 1

Exon 2 397 G>A V133I Missense SI Domain 1

Exon 2 401 T>C L134P* Missense SI Domain 1

Exon 2 403delG L134fs+101X* Frame shift SI Domain 1

Exon 2 430 A>G T144A Missense S1/S2 1

Exon 2 451_452delCT A150fs+132X Frame shift S2 Domain 1

Exon 2 458 C>T T153M* Missense S2 Domain 1

Intron 477+1 G>A Splice site S2 Domain 1 2

Intron 477+5 G>C Splice site S2 Domain 1 2

Intron 477+5 G>A Splice site S2 Domain 4 2

Exon 3 479 A>T El 60V* Missense/Splice S2 Domain 1

Exon 3 484 G>A V162M* Missense S2 Domain 1

Exon 3 488delT V162fs+73X Frame shift S2 Domain 1

Exon 3 502 G>A G168R Missense S2 Domain 15

Exon 3 502 G>C G168R Missense S2 Domain 4

Exon 3 504delG G168fs+67X* Frame shift S2 Domain 1

Exon 3 513 C>G Y171X Nonsense S2/S3 1

Exon 3 514 G>A V172M Missense S2/S3 2

Exon 3 520 C>T R174C Missense S2/S3 1

Exon 3 521 G>A R174H Missense S2/S3 1

Exon 3 524 534delTCTG R174fs+105X* Frame shift S2/S3 1 GTCCGCC

Exon 3 532 G>A A178T Missense S2/S3 1

Exon 3 535 G>A G179S Missense S2/S3 2

Exon 3 550 T>C Y184H* Missense S2/S3 1

Exon 3 556 G>C G186R* Missense S2/S3 1

Exon 3 564 G>A W188X* Nonsense S2/S3 1

Exon 3 569 G>A R190Q Missense S2/S3 3 Exon 3 569 G>T R190L* Missense S2/S3 1

Exon 3 573 577delGCGC L191fs+90X Frame shift S2/S3 4 T

Exon 3 583 OT R195W* Missense S2/S3 2

Exon 3 585delG R195fs+40X Frame shift S2/S3 4

Exon 3 592 A>G 1198V* Missense S3 Domain 1

Exon 3 595 T>G SI 99 A* Missense S3 Domain 1

Exon 3 604 G>A D202N Missense/Splice S3 Domain 1

Intron 605-2 A>G Splice site S3 Domain 1 3

Exon 4 612 OG I204M Missense S3 Domain 1

Exon 4 643 G>A V215M Missense S3 Domain 1

Exon 4 671 OT T224M* Missense S3/S4 1

Exon 4 674 OT S225L Missense S3/S4 8

Exon 5 691 OT R231C Missense S4 Domain 1

Exon 5 692 G>A R231H Missense S4 Domain 1

Exon 5 704 T>A 1235N Missense S4 Domain 2

Exon 5 722 T>G V241G* Missense S4 Domain 1

Exon 5 724 G>A D242N Missense S4 Domain 4

Exon 5 727delC D242fs+19X* Frame shift S4 Domain 1

Exon 5 727 OT R243C Missense S4 Domain 1

Exon 5 749 T>C L250P* Missense S4/S5 1

Exon 5 760 G>A V254M Missense S4/S5 10

Exon 5 775 OT R259C Missense S4/S5 5

Exon 5 776 780dupCCAC H258fs+5X* Frame shift S4/S5 1 C

Exon 5 776 G>T R259L Missense S4/S5 1

Exon 6 781 G>C E261Q* Missense/Splice S4/S5 1

Exon 6 781 G>T E261X* Nonsense/Splice S4/S5 1

Exon 6 784 OG L262V Missense S5 domain 1

Exon 6 796delC T265fs+22X Frame shift S5 domain 3

Exon 6 797 T>C L266P Missense S5 domain 30

Exon 6 803 T>G I268S* Missense S5 domain 1

Exon 6 805 G>A G269S Missense S5 domain 10

Exon 6 806 G>A G269D Missense S5 domain 4

Exon 6 815 G>A G272D Missense S5 domain 1

Exon 6 817 OT L273F Missense S5 domain 7

Exon 6 820 A>G I274V Missense S5 domain 1 Exon 6 829 T>C S277P* Missense S5 domain 1

Exon 6 830 OT S277L Missense S5 domain 2

Exon 6 839 T>A V280E Missense S5 domain 1

Exon 6 842 A>G Y281C Missense S5 domain 1

Exon 6 845 T>C L282P* Missense S5 domain 1

Exon 6 848 OG A283G* Missense S5/pore 2

Exon 6 862 880delGTGA A287fs+59X* Frame shift S5/pore 2 ACGAGTCAGGC CGCG

Exon 6 875 G>A G292D Missense S5/pore 1

Exon 6 877 OT R293C Missense S5/pore 4

Exon 6 905 C>T A302V Missense Pore 1

Exon 6 905 C>A A302E* Missense Pore 1

Exon 6 908 T>C L303P* Missense Pore 1

Exon 6 913 T>C W305R* Missense Pore 1

Exon 6 914 G>C W305S Missense Pore 1

Exon 6 914 G>A W305X Nonsense Pore 1

Exon 6 916 G>A G306R Missense Pore 1

Exon 6 916 G>C G306R Missense Pore 1

Exon 7 935 C>T T312I Missense Pore 2

Exon 7 940 G>A G314S Missense Pore 7

Exon 7 940 G>T G314C Missense Pore 1

Exon 7 944 A>G Y315C Missense Pore 4

Exon 7 947 G>T G316V* Missense Pore 1

Exon 7 958 C>T P320S* Missense Pore 1

Exon 7 964 A>G T322A Missense Pore/S6 2

Exon 7 965 C>T T322M Missense Pore/S6 4

Exon 7 973 G>A G325R Missense Pore/S6 6

Exon 7 1016 T>A F339Y* Missense S6 1

Exon 7 1017_1019delCTT 340delF In-frame del S6 1

Exon 7 1022 C>A A341E Missense S6 4

Exon 7 1022 C>T A341V Missense S6 8

Exon 7 1022 C>G A341G* Missense S6 1

Exon 7 1024 C>T L342F Missense S6 2

Exon 7 1028 C>T P343L Missense S6 1

Exon 7 1031 C>T A344V Missense/Splice S6 1

Exon 7 1032 G>A A344A Splice site S6 10

Intron 1032+1 G>T Splice site S6 1 7

Intron 1032+1 G>A Splice site S6 2 7

Intron 1032+2 T>C Splice site S6

7 ¹

Intron 1032+5 G>T Splice site S6

7 ¹

Exon 8 1046 OA S349X Nonsense C-Terminal ¹

Exon 8 1048 G>A G350R Missense C-Terminal 1

Exon 8 1052 T>C F351 S Missense C-Terminal 1

Exon 8 1061 A>G K354R* Missense C-Terminal ¹

Exon 8 1066 OT Q356X Nonsense C-Terminal 1

Exon 8 1075 OT Q359X* Nonsense C-Terminal 4

Exon 8 1079 G>T R360M* Missense C-Terminal 2

Exon 8 1085 A>G 362R Missense C-Terminal 5

Exon 8 1093 A>C N365H* Missense C-Terminal 1

Exon 8 1096 OT R366W Missense C-Terminal 8

Exon 8 1097 G>A R366Q Missense C-Terminal 1

Exon 8 1121 T>A L374H Missense C-Terminal 1

Intron 1128+1 G>A Splice site C-Terminal 1 8

Intron 1128+1 G>T Splice site C-Terminal 1 8

Intron 1128+5 G>A Splice site C-Terminal 1 8

Exon 9 1135 T>G W379G* Missense C-Terminal 1

Exon 9 1153 G>A E385 * Missense C-Terminal 1

Exon 9 1165 T>C S389P* Missense C-Terminal 1

Exon 9 1171_1173dupCTT 391dupS* In-frame ins C-Terminal 1

Exon 9 1177 1179dupTG 393dupW* In-frame ins C-Terminal 3 G

Exon 9 1189 OT R397W Missense C-Terminal 3

Exon 9 1193 A>G 398R* Missense C-Terminal 1

Exon 9 1196 1197delCCin 398fs+19X* Frame shift C-Terminal 1 sA

Exon 9 1202insC P400fs+61X Frame shift C-Terminal 1

Intron 1251+2 T>C Splice site C-Terminal 1 9

Exon 1265delA 421fs+9X* Frame shift C-Terminal 2 10

Exon 1338 OG D446E* Missense C-Terminal 2 10

Exon 1343 OT P448L* Missense C-Terminal 1 10

Exon 1351 OT R451W* Missense C-Terminal 1 10

Exon 1378 G>A G460S Missense C-Terminal 1 10

Exon 1430 OT P477L* Missense C-Terminal 1 11

Exon 1462delG E487fs+9X* Frame shift C-Terminal 1 11

Exon 1486_1487delCT T495fs+18X Frame shift C-Terminal 1 11

Exon 1513 OT Q505X* Nonsense/Splice C-Terminal 1 11

Intron 1515-2 del AG Splice site C-Terminal 1 11

Exon 1531 OT R511W* Missense C-Terminal 1 12

Exon 1552 OT R518X Nonsense C-Terminal 24 12

Exon 1553 G>A R518Q* Missense C-Terminal 1 12

Exon 1559 T>G M520R Missense C-Terminal 3 12

Exon 1565 A>C Y522S* Missense C-Terminal 1 12

Exon 1571 T>G V524G Missense C-Terminal 1 12

Exon 1573 G>A A525T Missense C-Terminal 1 12

Exon 1574 OT A525V Missense C-Terminal 1 12

Exon 1588 OT Q530X Nonsense C-Terminal 10 12

Exon 1591 OT Q531X* Nonsense/Splice C-Terminal 1 13

Exon 1597 OT R533W Missense C-Terminal 2 13

Exon 1615 OT R539W Missense C-Terminal 6 13

Exon 1616 G>A R539Q* Missense C-Terminal 1 13

Exon 1621 G>A V541I* Missense C-Terminal 1 13

Exon 1627 G>A E543 * Missense C-Terminal 1 13

Exon 1637 OT S546L Missense C-Terminal 4 13

Exon 1640 A>G Q547R* Missense C-Terminal 1 13

Exon 1663 OA R555S* Missense C-Terminal 1 13 Exon 1663 OT R555C Missense C-Terminal 4 13

Exon 1664 G>A R555H Missense C-Terminal 1 13

Exon 1669 A>G 557E Missense C-Terminal 1 13

Intron 1686-1 G>T Splice site C-Terminal 1 13

Exon 1696 T>C S566P* Missense C-Terminal 1 14

Exon 1697 OT S566F Missense C-Terminal 5 14

Exon 1697 OA S566Y Missense C-Terminal 2 14

Exon 1700 T>C I567T Missense C-Terminal 3 14

Exon 1702 G>A G568R Missense C-Terminal 7 14

Exon 1705 A>G 569E* Missense C-Terminal 1 14

Exon 1712 OT S571L* Missense C-Terminal 1 14

Exon 1760 OT T587M Missense C-Terminal 2 15

Exon 1766 G>A G589D Missense SAR 1 15

Exon 1771 OT R591C Missense SAR 1 15

Exon 1772 G>A R591H Missense SAR 7 15

Exon 1781 G>A R594Q Missense SAR 15 15

Exon 1781 G>C R594P Missense SAR 1 15

Exon 1786_1788delAGA 596delE* In-frame del SAR 1 15

Exon 1786 G>A E596 * Missense SAR 1 15

Exon 1794 G>A 598 * Splice site SAR 2 15

Intron 1794+1 G>T Splice site SAR 1 15

Exon 1799 OT T600M Missense SAR 3 16

Exon 1811insC D603fs+47X* Frame shift SAR 1 16

Exon 1831 G>A D611N* Missense SAR 1 16

Exon 1842_1844delCCA 614delH* In-frame del SAR 1 16

Exon 1876 G>A G626S Missense C-Terminal 1 16 Exon 1894insC P631fs+19X Frame shift C-Terminal 1 16

Exon 1903 G>A G635R* Missense C-Terminal 2 16

Exon 1986 OG Y662X* Nonsense C-Terminal 1 16

* denotes a novel variant, unique to this cohort. Deletion variants are indicated as del, insertions as ins, duplications as dup, and frameshift mutations are annotated for example as R174fs+105X format, where R174 represents the last properly encoded amino acid followed by a frameshift (fs) in the coding sequence resulting in 105 miscoded amino acids leading up to a premature stop codon (X). SAR = subunit assembly region.

Any appropriate method can be used to assess the cis or trans relationship of KCNQl mutations, KCNQl UTR variation, or wild-type sequences. For example, cDNA generated from KCNQ l niRNA (e.g., KCNQl cDNA generated by RT-PCR) can be sequenced to determine the cis or trans relationship of KCNQl mutations, KCNQl UTR variations, or wild-type sequences. Any appropriate biological sample such as a blood lymphocytes, skin biopsy, or myocardial biopsy can be used as a source for KCNQ l mRNA.

In some cases, a person identified as having a more severe form of LQTS can be at an increased risk for sudden death. In such cases, the person can be treated prophylactically with, for example, beta blocker therapy, ICD implantation, and/or sympathetic denervation.

In some cases, a human determined to have a cis or trans relationship of KCNQ l mutations, KCNQl UTR mutations, or wild-type sequences indicative of more severe LQTS can be treated with one or more microRNA inhibitors (e.g., anti-miRs) designed to reduce the ability of a microRNA (e.g., miR-378) to inhibit the expression of a wild-type

KCNQ 1 allele. A microRNA inhibitor can be designed to inhibit or reduce the activity of a particular microRNA (e.g., a miR-378). Examples of anti-miRs designed to inhibit or reduce the activity of miR-378 include, without limitation, cholesterol-based antagomiR agents and locked nucleic acid-based anti-miRs. In some cases, an anti-miR can have various backbone and/or sugar modifications such as 2'-0-methyl (2'-OMe), 2'-fluoro (2'

-F), phosphothioate linkages, and/or cholesterol conjugations.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

Example 1 - Functional variants in KCNQl 's 3 'UTR modify disease severity in patients with Type 1 LOTS through miR A-dependent mechanisms Genetic Analysis

LQT1 -associated KCNQ1 mutations were identified previously using standard protocols (Bhuiyan et ah, Prog. Biophys. Mol. Biol, 98(2-3):319-327 (2008)). For this analysis, the final exon and the 3'UTR of KCNQ1 were sequenced. The region to be analyzed included 3 amplicons and was amplified with polymerase chain reaction using oligonucleotide pairs: 5'-GGCACCTTCCCTTCTCTGG-3' (SEQ ID NO: 1) with 5'-ACC- ACCATGCCAGTGATGTC-3 ' (SEQ ID NO:2); 5'-CACAGCCTGCACTTGGG-3' (SEQ ID NO:3) with 5'-CAGGGCTCCTCTCCAGC-3' (SEQ ID NO:4); and 5'-CAGTCTCA- CCATTTCCCCAG-3' (SEQ ID NO: 5) with 5 '-GCCCAGAACAGGAGCGAC -3' (SEQ ID NO: 6).

In Siiico Analyses of Interactions Between miRNA and KCNQ1 3 'UTR SNPs

The KCNQ1 3 'UTR mRNA sequence was retrieved from the Ensemble Genome Browser (NCBI build 36, hgl 8), and SNPs identified experimentally in a KCNQ1 mutation carrier were mapped on the KCNQ 1 mRNA sequence. For each SNP, two sequences centered on the major or minor allele with 30 nucleotides flanking each side were assembled. The search by sequence option of www.mirbase.org was used to identify potential miRNA binding sites within these sequences using an E-value cut-off of 1000. This option used the miRanda algorithm to predict targeting of all miRNAs in the database to the input sequence. Comparison of the major and minor allele for miRNA target site predictions for each SNP was used to determine potential miRNA target sites that were created by the presence of the minor allele. miRNA Expression in Human Hearts

Post-mortem myocardial samples were obtained from subjects who had died due to non-cardiac causes. Total RNA was extracted using Trizol (Invitrogen), and real-time PCR was used to assess expression levels of miR-378, miR-21, miR-208b, and miR-133. miRNA expression was normalized for expression of U6 to evaluate the relative myocardial expression levels.

Luciferase Reporter Assay - Plasmid Construction

Using polymerase chain reaction, a luciferase reporter plasmid was constructed by amplifying the 3'UTR of KCNQ1 from a patient heterozygous for the SNPs rs2519184, rs8234, and rsl0798 with the following primers: 5 ' -ACTGACTAGTCATGGACC- ATGCTGTCTG-3 ' (SEQ ID NO: 7) and 5 ' -ACTGGAGCTCCAGCCTGTGATTCTC- CACG-3' (SEQ ID NO: 8). This 878 base pair fragment was cloned into the pMIR- REPORT™ Luciferase vector (Ambion) downstream of the luciferase coding region, creating luciferase-KCNQl-3'UTR-G-A-A (major haplotype) and luciferase-KCNQ 1 - 3'UTR-A-G-G (minor haplotype). The miR-378 overexpression vector, pCDHl-miR- 378, was constructed by PCR-amplification of miR-378 precursor DNA form human genomic DNA using the following primers: 5 '-ACTGGAATTCAGAAAGAGGCTG- CGAGGAG-3 ' (SEQ ID NO: 9) and 5 ' -ACTGGGATCCGGAACAACCAGAAC- ATCTCAC-3' (SEQ ID NO: 10). This 306 base pair fragment was cloned into the pCDHl-MCSl-EFl-Puro vector (System Biosciences) under the control of a CMV promotor. The negative control miRNA overexpression vector, PCDHl-control-miRNA, was based on the pcDNATM6.2-GW/miR-neg control plasmid (Invitrogen), which contained an insert that can form a hairpin structure that is processed into mature miRNA but is predicted not to target any known vertebrate gene. All generated constructs were verified by sequencing.

Cell Isolation and Preparation

Neonatal rat cardiac myocytes, immortalized with a temperature-sensitive SV40 T antigen (H10 cells) as described elsewhere (Jahn et ah, J. Cell Set, 109(Pt 2):397-407 (1996)), were cultured in Dulbecco's Modified Eagles Medium supplemented with 10% fetal calf serum (Gibco-BRL) and glutamine at 33 °C. One day prior to transfection, cells were seeded in a 24-well plate at a density of 1.4xl0⁶ cells per plate. Cardiomyocytes from 1-2-day-old Lewis neonatal rats were isolated and cultured as described elsewhere (Leenders et al, J. Biol. Chem., 285(35):27449-27456 (2010)). Transfection and Luciferase Assay

H10 cells were transiently transfected per well with 5 ng Renilla luciferase plasmid, phRL vector (Promega), and 5 ng or 10 ng of either luciferase-KCNQ 1 -3 'UTR- G-A-A, luciferase-KCNQ 1 -3 'UTR-A-A-A, luciferase-KCNQ 1 -3 'UTR-G-G-A, or luciferase-KCNQ 1 -3 'UTR-A-G-G using GeneJammer (Agilent Technologies). Neonatal rat cardiomyocytes were transfected with 50 ng Renilla luciferase plasmid, phRL vector, and 100 ng of either luciferase-KCNQ 1 -3 'UTR-G-A-A, luciferase-KCNQ 1-3 'UTR-A-A- A, luciferase-KCNQ 1-3 'UTR-G-G-A, or luciferase-KCNQ 1-3 'UTR-A-G-G using lipofectamine 2000 reagent (Invitrogen). COS-7 cells were transiently transfected per well with 5 ng Renilla luciferase plasmid, phRL vector, 100 ng of either luciferase- KCNQ 1-3 'UTR-G-A-A or luciferase-KCNQ 1-3 'UTR-A-G-G, 100 ng of miR-378 or negative control miRNA overexpression construct, and empty pCDHl as filler using polyethylenimine (PEI). At 48 hours after transfection, cells were lysed and assayed for luciferase and Renilla luciferase activity with a luminometer (Glomax multi detection system, Promega) by using the Renilla reporter assay system (Promega). Renilla luciferase activity was assayed to normalize luciferase results for cell densities and transfection efficiency. Statistical Methods

QTc was calculated using Bazett's formula and compared between the various LQT1 genotype and 3 'UTR SNP haplotype combinations. A linear mixed effect regression model from the kinship package in R was used (R Development Core Team. R: a language and environment for statistical computing. The R Project for Statistical Computing Web Site. (World Wide Web at "r-proj ect.org") (2009)). Differences in symptom prevalence were analyzed with a logistic regression model using generalized estimation equations. In both models, a correction for the relatedness among individuals was applied. Furthermore, age at the time of ECG, gender, and proband status were included as covariates, and genotype effects were modeled as additive effects, p-values <0.05 was regarded as statistically significant. Results

SNPs are present in the 3 'UTR of KCNQl

From two institutions (Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands (AMC) and Mayo Clinic, Rochester, Minnesota, USA (MC)), families were included where a mutation in KCNQl was identified in at least three members, and at least one affected member displayed QTc prolongation. Individuals with acquired cardiac diseases, electrolyte abnormalities, or use of medications known to prolong repolarization were excluded. From the AMC, 84 LQT 1 -positive subjects from 24 families were included. From the MC, 84 LQT 1 -positive subjects from 17 families were included. Informed written consent was obtained from all subjects and demographic data (Table 1) and details of the identified mutations (Table 2) were collected. In all subjects, the final exon and the complete 3 'UTR of KCNQl were sequenced. In the AMC cohort, seven SNPs were found of which six SNPs were in the 3 'UTR. SNPs at nucleotide positions 403092 and 403641 were not reported previously (NCBI build 36, hgl8). Except for the SNP at nucleotide position 403092, all SNPs (and no additional SNPs) were also found in the MC population (Figure 1A; Table 3 for SNP frequencies).

Table 1. Clinical characteristics, mutation types, and ECG parameters of the study

Mutation type

Missense, n (%) 63 (75) 67 (80)

Frameshift, n (%) 7 (8) 1 (1)

Splice site, n (%) 1 1 (13) 4 (5)

Deletion, n (%) 3 (4) 12 (14)

ECG parameters

RR interval (msec) 859±19 917±24

Males 879±26 968±41

Females 844±28 875±27

QT duration (msec) 411±7 426±6

Males 405±8 429±10

Females 416±10 425±8

QTc (msec) 446±7 451 ±4

Males 435±6 445±6

Females 454±7* 457±4*

Values are expressed as number of patients (percentage of total), or as mean ± standard error of the mean (SEM). * denotes statistical significance compared to males.

Table 2. Mutations in KCNQ1 found in the study populations from AMC and MC.

Region Nucleotide change Mutation Mutation type Location Cr. # Ref.

Exon 1 360 G>C W120C Missense N-terminus A 2 1

Exon 1 365 G>A C122Y Missense N-terminus M 1 2

Exon 2 387-?_1393+?del Deletion SI A 3 -

Intron 2 477+1 G>C Splice site S2 A 3 3

Exon 3 551 A>C Y184S Missense S2/S3 A 7 4

Exon 3 739 T>C F193L Missense S2-S3 A 1 5*

Exon 4 674 OT S225L Missense S3/S4 A, M 1, 3 6* Exon 5 704 T>A I235N Missense S4 M 20 ₇*

Exon 5 727 OT R243C Missense S4 A, M 14, 3 8*

Exon 5 775 OT R259C Missense S4/S5 A 2 9*

Exon 5 776 G>T R259L Missense S4/S5 M 1 2

Exon 6 797 T>C L266P Missense S5 M 3 10

Exon 6 805 G>A G269S Missense S5 M 3 11 *

Exon 6 806 G>A G269D Missense S5 M 2 12*

Exon 6 820 A>G I274V Missense S5 A 4 1

Exon 6 875 G>A G292D Missense S5/pore A 2 1

Exon 6 887 T>C F296S Missense S5/pore A 9 13*

Exon 7 940 G>A G314S Missense Pore A 1 14*

Exon 7 941 G>A G314D Missense Pore A 1 15

Exon 7 944 A>G Y315C Missense Pore M 7 6*

Exon 7 964 A>G T322A Missense Pore/S6 M 9 15

Exon 7 1015_1017delTTC 339delF Deletion S6 M 12 16*

Exon 7 1031 OT A344V Missense S6 A 4 17*

Intron 7 1032+5 G>A Splice site S6 A, M 8, 4 18*

Exon 9 1189 OT 397W Missense C-terminus A 6 1

Exon 10 1265 A>C 422T Missense C -terminus A 3 19

Exon 10 1343 ins C P448fsX13 Frameshift C-terminus A, M 7, 1 -

Exon 12 1571 T>G V524G Missense C-terminus M 2 1

Exon 13 1615 OT R539W Missense C-terminus M 4 20*

Exon 14 1700 T>G I567S Missense C-terminus M 4 15

Exon 15 1771 OT R591C Missense SAD A 2 1

Exon 15 1772 G>A R591H Missense SAD M 5 21 *

Exon 15 1781 G>A R594Q Missense SAD A 4 22*

Del, deletion; ins, insertions; P448fsX13, frameshift mutation whereby P448 represents the last normally encoded amino acid followed by a frameshift (fs) in the coding sequence generating 13 miscoded amino acids leading up to a premature stop codon (X); 387- ?_1393+?del, mutation leading to the deletion of exon 2 (starting from nucleotide 387) to exon 10 (ending at nucleotide 1393), but whereby the exact nucleotide change is not yet determined (?); N-terminus, amino-terminus; S, transmembrane segment; C-terminus, carboxyl-terminus; SAD, subunit assembly domain; Cr., center in which the mutation was found (A: Academic Medical Center Amsterdam, M: Mayo Clinic); #, number of individuals within each study population affected with the mutation; Ref, reference linking the mutation with long QT syndrome; *, biophysical/loss-of-function properties of the mutation are described in the reference.

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9. Kubota et al., J. Cardiovasc. ElectrophysioL, 1 1(9): 1048-1054 (2000).

10. Splawski et al., Circulation, 102( 10): 1 178- 1 185 (2000).

11. Murray et al., J. Med. Genet., 39(9):681-685 (2002).

12. Chouabe et al, EMBO J., 16(17):5472-5479 (1997).

13. Yang et al, Circ. Arrhythm ElectrophysioL, 2(4):417-426 (2009).

14. Li et al., Biochem. Biophys. Res. Commun., 383(2):206-209 (2009).

15. Choi et al., Circulation, 1 10(15):2119-2124 (2004).

16. Thomas et al., Cardiovasc. Res., 67(3):487-497 (2005).

17. Siebrands et al., Anesthesiology, 105(3):511-520 (2006).

18. Tsuji et al., J. Mol. Cell. Cardiol., 42(3):662-669 (2007).

19. Itoh et al., Heart Rhythm., 7(10): 141 1-1418 (2010).

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21. Grunnet et al, Heart Rhythm., 2(1 1): 1238-1249 (2005).

22. Huang et al., Cardiovasc Res., 51 (4):670-680 (2001).

Table 3. Single nucleotide polymorphisms found in the 3 ' untranslated region oiKCNQl.

AMC MC

(n = 84) = 84)

Genetic Nucleotide Allele SNP ID Genotype N (%) Minor N (%) Minor

Location change (Major/ allele allele position minor) frequency frequency

Exon 16 402865 C/T rsl 1601907 CC 38 (45) 0.327 50 (60) 0.214

CT 37 (44) 32 (38)

TT 9 (11) 2 (2)

3'UT 403092 G/A not available GG 81 (96) 0.018 84 (100) 0.000

GA 3 (4) 0

AA 0 0

3'UTR 403321 C/T rs45460605 CC 81 (96) 0.018 83 (99) 0.006

CT 3 (4) 1 (1)

TT 0 0

3'UTR 403389 G/A rs2519184 GG 78 (93) 0.036 50 (60) 0.202

GA 6 (7) 34 (40)

AA 0 0 3 'UTR 403641 G/A not available GG 83 (99) 0.006 83 (99) 0.006

GA 1 (1) 1 (1)

AA 0 0

3 'UTR 403785 A/G rs8234 AA 52 (62) 0.208 31 (37) 0.351

AG 29 (34) 47 (56)

GG 3 (4) 6 (7)

3 'UTR 403842 A/G rsl0798 AA 52 (62) 0.208 31 (37) 0.351

AG 29 (34) 47 (56)

GG 3 (4) 6 (7)

Position of the nucleotide change is starting from the ATG start codon ( CBI build 36, hgl8). The SNP ID denotes single nucleotide polymorphism identity from public

databases. N (%) denotes number of patients (percentage of total). Minor variants of SNPs rs2519184 and rs8234 in KCNQ1 's 3 'UTR create binding sitesor miRNA-378

The mirbase algorithm (World Wide Web at "mirbase.org") predicted that the minor (A) variant of SNP rs2519184 and the minor (G) variant of SNP rs8234 create binding sites for a number of miRNAs including two binding sites for miRNA-378 (miR- 378; Figure IB). miR-378 was found to be abundantly expressed in the human left

ventricle of four healthy donor hearts, at levels comparable to miR-208b (miR-208b is expressed in heart tissue and a low levels in skeletal muscle) (Figure 1C).

Minor variants of SNPs rs2519184 and rs8234 repress mRNA expression in the presence of miR-378

To assess the functional effects of SNPs rs2519184 and rs8234 on mRNA

expression, the 3 'UTR oiKCNQl were cloned with either the major 3'UTR SNP

haplotype (G-A-A) or the minor haplotype (A-G-G) of SNPs rs2519184, rs8234 including rsl0798 as the latter two SNPs were in complete linkage disequilibrium in all patients in this study. Next, these 3 'UTRs were placed downstream of the luciferase coding region in the pMIR-REPORT™ plasmid (Ambion) and transfected into both heart-derived H10 cells (Figure 2A) and primary neonatal rat cardiomyocytes (Figure 6A). The plasmid containing the minor 3 'UTR SNP haplotype (A-G-G), which harbors two created binding sites for miR-378, had significantly lower luciferase activity in both cardiac cell types. Moreover, introduction of either minor allele of SNP rs2519184 or rs8234 into the major allele was sufficient to decrease luciferase activity, indicating that those two nucleotides control translation oiKCNQl (Figure 2B for H10 cells, and Figure 6B for neonatal rat cardiomyocytes). To assess the specific effect of miR-378 on mRNA expression, miR- 378 was overexpressed in COS-7 cells transfected with a reporter containing either the major 3'UTR SNP haplotype (G-A-A) or the minor SNP haplotype (A-G-G).

Overexpression of miR-378, but not of a control miRNA, repressed luciferase activity from the reporter containing the minor 3 'UTR SNP haplotype (A-G-G), but not from the reporter containing the major SNP haplotype (G-A-A; Figure 2C).

Inhibition of microRNA-378 by a specific anti-miR solely directed against microRNA-378 inhibited this microRNA and abolished the suppressive effects of the SNPs (Figures 8 and 9).

The experiment of Figure 9 was repeated several times, and the combined results are presented in Figure 10.

An additional experiment was performed to show the allelic imbalance on mRNA levels, thereby confirming that there is imbalance on both the polypeptide and mRNA levels. The allelic imbalance on mRNA level was demonstrated using an allele-specific qPCR approach. In this experiment, primers were designed to specifically amplify and detect the KCNQ1 3'UTR depending on the variant in the 3 'UTR. Briefly, for the allele- specific qPCR, 400 ng of input RNA was used in the reverse transcription reaction. Input RNA was reverse transcribed using superscript II Reverse transcriptase (Invitrogen) with oligo-dT primers according to the manufacturer's protocol.

The quantitative PCR was performed using LightCycler 480 sybr green I master (Roche). For SNP rs8234, allele-specific reverse primers (5'-ACCACAAAT- TATTGATTTCTATGCGAT-3 ' (SEQ ID NO: 1 1) for amplifying the major A allele or 5 ' -ACCACAAATTATTGATTTCTATGCGAC-3 ' (SEQ ID NO: 12) for amplifying the minor G allele) and a general forward primer (5'-AGCCAGCCAAACACACAG-3 ' (SEQ ID NO: 13)) were used. Quantitative PCR reactions were performed on a HghtCycler480 system II (Roche) using the following program: 5 minutes pre-incubation at 95°C and 40 cycles of 10 seconds of denaturation at 95°C, 20 seconds of annealing at 60°C, and 20 seconds of elongation at 72°C. Data were analyzed using LinRegPCR quantitative PCR data analysis software. The starting concentrations of transcripts estimated by this software were corrected for the estimated starting concentrations of the housekeeping gene GAPDH (5'-ACCCACTCCTCCACCTTTGAC-3' (SEQ ID NO: 14) and 5'- ACCCTGTTGCTGTAGCCAAATT-3 ' (SEQ ID NO: 15)) and for the estimated starting concentration of a total KCNQ1 amplicon closely resembling the allele-specific amplicons (5 ' -GAAGTGACGGTTCCTACAC-3 ' (SEQ ID NO: 16) and 5'- AGCTTGCACAATTAATAATCAAAATC-3 ' (SEQ ID NO: 17)).

By directly comparing the amplification of the major and minor variant, it was possible to calculate the relative expression. A 10% difference in expression between major and minor allele was detected, where the minor allele was less abundant (Figure 11). This demonstrates that the minor variants were suppressive variants.

In LQT1, SNPs rs2519184 and rs8234 modify QTc in an allele-specific fashion

Since the functional analysis revealed that the minor alleles of SNPs rs2519184 and rs8234 suppress translation, it was hypothesized that this translational suppression would increase or decrease QTc depending on whether they occur in trans (opposite allele) or in cis (on the same allele) to the LQT1 -causative KCNQ1 mutation, as defined by analysis of phase in the families. Occurrence of 'suppressive 3 'UTR SNPs' in cis to the LQT 1 mutation would be expected to attenuate QTc prolongation by decreasing the abundance of mutant protein available for tetrameric assembly, while 'suppressive 3'UTR SNPs' in trans to the mutation would have the opposite effects by decreasing translation of the normal allele and thereby increasing the amount of mutant K_v7.1 sub-units resulting in greater QTc prolongation (Figure 7). The statistical approach to assess differences in QTc is described herein.

The presence of the suppressive minor (A) allele of SNP rs2519184 in trans to the mutated allele (i.e., on the normal allele) was associated with a marked increase in QTc by 46 ± 16 milliseconds in the initial AMC cohort (Figure 3 A). This was corroborated by similar findings in the MC cohort where the presence of the minor allele in trans was associated with 60 ± 16 milliseconds longer QTc (Figure 3D). Notably, in the MC cohort, subjects were also identified where this 3 'UTR minor allele SNP resided on the LQTl-mutation containing allele (i.e., in cis to the mutation). Here, location of this SNP in cis was associated with 16 ± 8 milliseconds shorter QTc. The same effects were seen for the suppressive SNP rs8234. When the minor SNP rs8234 (G) was in trans to the mutated allele, QTc was increased by 26 ± 10 milliseconds (Figure 3B). Again, this was confirmed in the MC cohort where G located in trans increased the QTc by 33 ± 7 milliseconds (Figure 3E). The opposite location of these suppressive minor alleles (i.e., in cis to the mutation) was related with shorter QTc (minus 41 ± 25 milliseconds), which was confirmed in the MC population (reduction by 9 ± 9 milliseconds; not significant).

Allele-specific haplotype analysis of SNPs rs2519184 and rs8234 revealed that location of the suppressive minor alleles of the SNPs (A-G) in trans to the mutation was associated with 49 ± 16 milliseconds longer QTc in the AMC cohort and a 60 ± 14 milliseconds longer QTc in the MC cohort (Figure 3C and Figure 3F). Subjects where the A-G-G haplotype was in cis to the mutation were only present in the MC cohort. Indeed, in these subjects, the opposite effect on QTc was suggested, as the QTc was now attenuated by 12 ± 9 milliseconds. Various allele-specific haplotype combinations of KCNQ1 and the minor alleles of the three SNPs, as shown in Figure 3C and Figure 3f, displayed an intermediate effect on QTc.

SNPs rs2519184 and rs8234 modify QTc in an allele-specific fashion in singles LQT1 families

The data provided herein demonstrates that testing for the suppressive 3 'UTR

SNPs can predict disease severity of a given KCNQ1 mutation in one single family. To test this further, the allele-specific haplotype of SNPs rs2519184 and rs8234 with regard to QTc in the largest families in the AMC (14 affected) and MC (20 affected) cohorts were analyzed. Indeed, the KCNQ1-R243C carriers, where the minor suppressive alleles of one or two SNPs was found in trans to the mutation, exhibited markedly longer QTc (533 ± 24 and 489 ± 25 milliseconds, respectively) than their KCNQl-R243C-positive relatives who did not carry these SNPs (441 ± 7 milliseconds; Figure 4A). The opposite was seen in the largest MC family where the 3 'UTR SNPs resided on the mutation (KCNQl-I235N)-containing allele. Family members with the suppressive SNPs in cis to the mutation displayed shorter QTc values (421 ± 6 milliseconds) than family members lacking these SNPs (458 ± 17; Figure 4B). In LQT1, SNPs rs2519184 and rs8234 modify symptomatology in an allele-specific fashion

The following was performed to assess whether allele-specific location of these suppressive SNPs in the KCNQFs 3'UTR also modifies the known cardiac sequelae of LQT1 (unexplained syncope, documented torsades de pointes ventricular

tachycardia/ventricular fibrillation, and/or [aborted] sudden death). Location of the suppressive allele of SNPs rs8234 in trans to the mutation was associated with a statistically significant increased occurrence of symptoms. Inversely, the opposite location of the minor suppressive SNP haplotype (A-G) (i.e., in cis to the mutation) tended to be related to fewer symptoms (Figure 5).

The effect of SNPs rs2519184, rs8234, and rs 10798 on QTc in the general population To study the effect of SNPs rs2519184, rs8234, and rs l0798 in the general population, the following was performed using the KORA Study to evaluate whether the SNPs rs2519184, rs8234, or rs 10798 are correlated with QTc duration in the general population. The KORA Study is a series of independent population-based

epidemiological surveys of participants living in the region of Augsburg, Southern Germany. All survey participants were residents of German nationality identified through the registration office and were examined in 1994-95 (KORA S3) and 1999-2001 (KORA S4). In 2004-05, 3.006 subjects participated in a 10-year follow-up examination of S3 (KORA F3) and in 2006-08, 3.080 subjects participated in a 7-year follow-up examination of S4 (KORA F4). Individuals for genotyping in KORA F3 and KORA F4 were randomly selected.

The data about rs2519184 is described elsewhere (Pfeufer et ah, Circ. Res.,

96(6):693-701 (2005)). A correlation was found between the SNP and QTc duration in screening sample of 689 individuals, but this association was not found in a confirmation sample of 3277 individuals. An analysis of KORA reveals that rs8234 and rs 10798 were not associated with QTc duration in two populations of the KORA study (Table 4), suggesting that the general suppressive effects of these SNPs does not lower KCNQ1 proteins sufficiently to induce QTc prolongation in healthy individuals. However, as indicated herein, effects of the suppressive 3 'UTR SNPs were pronounced when the two KCNQ1 alleles harbours a disease-causing mutation.

Table 4. The effect of SNPs rs8234 and rsl0798 on QTc in the general population.

Major Minor Effect on QTc

SNP Population* Number allele/haplotype allele/haplotype MAF Beta (msec) p- value rs8234 KORA F3 1459 A G 0.3041 -0.573105 0.52 rsl0798 KORA F3 1459 A G 0.304 -0.572674 0.52 rs8234 KORA S4 975 A G 0.3216 1.383768 0.13 rsl0798 KORA S4 975 A G 0.3209 1.42896 0.12

Haplot pe KORA F3 1459 AA GG 0.3047 0.569323 0.52

Haplot pe KORA S4 975 AA GG 0.3185 0.611701 0.42 Number: number of individuals within the screening sample; MAF: minor allele frequency. The effect on the QTc duration refers to the dosage (i.e., expected number of copies) of the major allele. The QTc duration is calculated by the Bazett's formula, and is corrected for age and gender. The results provided herein demonstrate that SNPs in the 3 'UTR of KCNQ1

(SNPs rs2519184 and rs8234) create binding sites for miR-378, a miRNA appreciably expressed in the human heart. Creation of these binding sites can allow miR-378 to suppress translation of the SNP-containing KCNQ1 allele. These results are clinically relevant when these suppressive 3 'UTR SNPs occur in carriers of an LQT1 -causing mutation. The allelic location of these suppressive SNPs can alter the balance between normal and mutated K_v7.1 channel subunits. When the SNPs are in cis to the mutation, the diseased allele can be suppressed and the expressed LQT1 phenotype can be less severe. However, when the suppressive SNPs reside on the normal non-mutated allele (in trans), then translation of the normal allele can be suppressed and the LQT1 phenotype can be more severe.

The results provided herein also demonstrate that LQT1 genetic testing can include an analysis and cis/trans phase determination of the suppressive 3 'UTR SNPs in KCNQ 1. More generally, the results provided herein demonstrate that genetic variation in the 3 'UTR may be an important source for clinical variability by altering the balance of translation between the two alleles. It is noteworthy that the size of the effects on QTc duration and symptoms that is described go well beyond what has been shown for modifiers described elsewhere (Pfeufer et ah, Circ. Res., 96(6):693-701 (2005); and Crotti et ah, Circulation,

120(17): 1657-1663 (2009)).

In summary, the results provided herein demonstrate that naturally occurring

SNPs in KCNQl 's 3'UTR suppress translation by creating binding sites for miR-378. In KCNQ1 -mutation carriers, these functional SNPs explain an important part of the incomplete penetrance and variable expressivity associated with LQT1, suggesting that the clinical effects of a pathogenic mutation may be determined by naturally occurring genetic variation in its 3 'UTR that results in the miRNA-mediated alteration of normal or mutant allele expression. These results also suggest that in autosomal dominant diseases like long QT syndrome, disease severity can be importantly modified even by sequence variation in the 3'UTR stemming from the unaffected parent. OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for assessing the cis or trans nature of a human heterozygous for a KCNQ 1 mutation and heterozygous for a KCNQ 1 UTR variation, wherein said method comprises:

(a) determining if an allele of said human comprises (i) said KCNQ 1 mutation and said KCNQ l UTR variation, (ii) a lack of said KCNQl mutation and a lack of said KCNQl UTR variation, (iii) said KCNQl mutation and a lack of said KCNQl UTR variation, or (iv) said KCNQ 1 UTR mutation and a lack of said KCNQ 1 variation,

(b) classifying said human as having said KCNQl mutation and said KCNQl

UTR variation in cis if said allele comprises (i) or (ii), and

(c) classifying said human as having said KCNQ l mutation and said KCNQl UTR variation in trans if said allele comprises (iii) or (iv).

2. The method of claim 1, wherein said human is an LQT1 patient.

3. The method of claim 1, wherein said allele comprises said KCNQl mutation and said KCNQ 1 UTR variation, and said human is classified as having said KCNQ 1 mutation and said KCNQl UTR variation in cis.

4. The method of claim 1, wherein said allele comprises a lack of said KCNQl mutation and a lack of said KCNQl UTR variation, and said human is classified as having said KCNQ l mutation and said KCNQl UTR variation in cis.

5. The method of claim 1, wherein said allele comprises said KCNQl mutation and a lack of said KCNQl UTR variation, and said human is classified as having said KCNQ l mutation and said KCNQl UTR variation in trans.

6. The method of claim 1 , wherein said allele comprises said KCNQ 1 UTR variation and a lack of said KCNQl mutation, and said human is classified as having said KCNQl mutation and said KCNQl UTR variation in trans.

7. The method of claim 1 , wherein said KCNQ 1 mutation is L266P, R518X, G 168R, or R594Q.

8. The method of claim 1 , wherein said KCNQ 1 UTR variation is an rs2519184 SNP or rs8234 SNP.

9. A method for treating a mammal having a KCNQl UTR variation that is part of a microRNA binding site, wherein the binding of a microRNA to said binding site reduces expression of a K_v7.1 potassium channel subunit lacking a mutation associated with LQTS, wherein said method comprises administering an miRNA inhibitor to said mammal under conditions wherein said miRNA inhibitor reduces the ability of said microRNA to reduce expression of said subunit.

10. The method of claim 9, wherein said mammal is a human.

11. A method for treating a human comprising administering an agent that inhibits or mimics the activity of a microRNA that targets a KCNQl UTR variation to increase the ratio of expression of a non-mutated KCNQl allele to mutated KCNQl allele.

12. A method for treating a human having long QT syndrome, wherein said method comprises:

(a) determining if an allele of said human comprises (i) said KCNQ 1 mutation and said KCNQ l UTR variation, (ii) a lack of said KCNQl mutation and a lack of said KCNQl UTR variation, (iii) said KCNQl mutation and a lack of said KCNQl UTR variation, or (iv) said KCNQ 1 UTR mutation and a lack of said KCNQ 1 variation, and (b) administering a beta blocker to said human, implanting an implantable cardioverter-defibrillator into said human, or performing a sympathetic denervation procedure on said human.

13. The method of claim 12, wherein said method comprises administering said beta blocker to said human.

14. The method of claim 13, wherein said beta blocker is metoprolol, carvedilol, bisoprolol, nebivolol, or propanolol.

15. The method of claim 12, wherein said method comprises implanting said implantable cardioverter-defibrillator into said human.

16. The method of claim 12, wherein said method comprises performing said sympathetic denervation procedure on said human.