KR19990076623A - Long-term QT syndrome gene encoding KVLQT1 and a combination of KVLQT1 and MINK - Google Patents

Abstract

One aspect of the invention relates to the identification of the molecular framework of long-term QT syndrome. More specifically, the present invention confirmed that mutated KVLQT1 causes long-term QT syndrome. Analysis of this gene will provide early diagnosis of subjects with long-term QT syndrome. This diagnostic method consists of analyzing the nucleic acid sequence of the KVLQT1 gene of a subject and comparing it with the nucleic acid sequence of a natural, unmutable gene. In addition, the amino acid sequence of KVLQT1 can be analyzed for mutations that cause long-term QT syndrome. Pre-diagnosis of long-term QT syndrome will enable doctors to treat the disease using current medical therapies. The second aspect of the present invention relates to the understanding that KVLQT1 associates with minK to form cardiac potassium channels. This allows the analysis of drugs interacting with these channels to identify new drugs useful for treating or preventing long-term QT.

Description

Long-term ＱＴ syndrome gene encoding ＫＶＬＱＴ1 and a combination of ＫＶＬＱＴ1 and ｉｉＫ

This application was made with government support under license number R01 HL48074, funded by the National Institutes of Health, Bethesda, Maryland, and license number M01 RR00064 of the Public Health Service.

The present invention relates to genes and gene products associated with long-term QT syndrome (LQT), and methods of diagnosing and preventing LQT. LQT is diagnosed by analyzing the DNA sequences of the subject's KVLQT1 gene and comparing each DNA sequence with a known DNA sequence of the normal KVLQT1 gene. In addition, the KVLQT1 gene of a subject can be screened for mutations that cause LQT. LQT's predictions will enable doctors to prevent this disease using current medical therapies. The present invention also relates to the discovery that KVLQT1 and minK proteins associate together to form cardiac I _Ks potassium channels. This knowledge can be used to co-express these two proteins in cells, and such transformed cells can be used for the screening of drugs useful for treating or preventing LQT.

The literature and other materials used herein to explain the background of the invention or to provide further details related to its practice are incorporated by reference and are, for convenience, classified into the accompanying references list.

Deep vein is a common cause of morbidity and mortality, responsible for about 11% of all natural deaths (Kannel, 1987; Willich et al., 1987). In general, prediagnosis and treatment of individuals with life-threatening ventricular tachyarrhythmias are insufficient, and in some cases medical treatment actually increases the risk of arrhythmia and death (New Engl. J. Med. 327, 227 (1992). Because of these factors, early detection of the risk of deep vein and prevention of arrhythmia are of great importance.

Both genetic and acquired factors contribute to the risk of developing deep veins. Long-term QT syndrome (LQT) is a genetic deep vein that causes sudden loss of consciousness, fainting, sudden seizures, and sudden death from ventricular tachyarrhythmias, especially tordes de pointes and ventricular fibrillation. (Ward, 1964; Romano, 1965; Schwartz et al., 1975; Moss et al., 1991). The disease generally develops in young, otherwise healthy individuals (Ward, 1964; Romano, 1965; Schwartz, 1975). Most LQT gene carriers show prolongation of the QT interval of the electrocardiogram, a signal of aberrant cardiac repolarization (Vincent et al., 1992). The clinical characteristics of LQT arise from repolarization-associated ventricular tachyarrhythmias such as Torsade de Pointes, which refers to the transient pulmonary arrhythmias, especially the characteristic pulsation of electrocardiograms in these arrhythmia and ventricular fibrillation (Schwartz et al., 1975: Moss and McDonald, 1970). Torsade de Pointes can be denatured into ventricular fibrillation, especially lethal arrhythmia. Although LQT is not a common diagnostic, ventricular arrhythmias are very common, with more than 300,000 US citizens dying every year (Kannel, et al., 1987; Willich et al., 1987), and in many cases the underlying mechanism. May be an ideal cardiac repolarization. Therefore, LQT offers the only opportunity to study life-threatening deep veins at the molecular level.

Both genetic and acquired forms of LQT have been defined. Acquired LQT and secondary arrhythmias can arise from cardiac ischemia, bradycardia and metabolic abnormalities such as low serum potassium or calcium concentrations (Zipes, 1987). LQT may also result from treatment with certain medications, including antibiotics, antihistamines, general anesthetics and most commonly antiarrhythmic medications (Zipes, 1987). Genetic LQT forms result from mutations in three or more different genes. In previous studies, the LQT locus was assigned to chromosomes 11p15.5 (LQT1) (Keating et al., 1991a; Keating et al., 1991b), 7q35-36 (LQT2) and 3p21-24 (LQT3) (Jiang et al., 1994). Of these, the most common cause of genetic LQT is LQT1. The data in this application indicate that at least 50% of the genetic LQTs are due to mutations in this gene (Q. Wang, closed results). Recently, the fourth LQT site (LQT4) was mapped to 4q25-27 (Schott et al., 1995). Applicants' studies indicate that minK, a gene located on chromosome 21, is also associated with LQT.

Autosomal dominant and autosomal recessive forms of this disease have been reported. Autosomal recessive LQT (also known as Jervell-Lange-Nielson syndrome) is associated with congenital neurodeafness, and this form of LQT is rare (Jervell and Lange-Nielson, 1957). Autosomal dominant LQT (Romano-Ward syndrome) is more common and is not associated with other phenotypic abnormalities. Diseases very similar to genetic LQT may generally be obtained as a result of pharmacological therapy (Schwartz et al., 1975; Zipes, 1987).

This data is related to the arrhythmia mechanism of LQT. Two hypotheses have already been proposed for LQT (Schwartz et al., 1994). One suggests that the preponderance of left autonomic nerve dominance causes abnormal cardiac repolarization and arrhythmia. This hypothesis is supported by the finding that arrhythmia in dogs can be induced by the removal of dominant ganglia. In addition, other evidence suggests that some LQT patients are effectively treated by β-adrenergic blockers and also by left stellate ganglion resection (Schwartz et al., 1994). A second hypothesis for LQT related arrhythmias suggests that mutations in the cardiac specific ion channel gene, or the gene that regulates the cardiac ion channel, cause delayed myocyte repolarization. Delayed myocyte repolarization can promote activation of L-type calcium channels, resulting in secondary depolarization (January and Riddle, 1989). This secondary depolarization is almost the same as the cellular mechanism of Torsade de Pointes arrhythmia (Surawicz, 1989). This hypothesis is supported by the observation that pharmacological blockade of potassium channels can induce QT prolongation and repolarization-related arrhythmias in human and animal models (Antzelevitch and Sicouri, 1994). The discovery that one form of LQT arises from mutations in the cardiac potassium channel gene supports the myocyte hypothesis.

In 1991, a complete link between autosomal dominant LQTs and polymorphs in HRAS was reported (Keating et al., 1991a; Keating et al., 1991b). This finding positioned LQT1 on chromosome 11p15.5 and allowed prediagnosis in some departments. Autosomal dominant LQT was previously thought to be genetically homologous, and the first seven families studied were linked to 11p15.5 (Keating et al., 1991b). In 1993, it was found that there was position heterogeneity for LQT (Benhorin et al., 1993; Curran et al., 1993; Towbin et al., 1994). Two additional LQT positions were later identified with LQT2 on chromosome 7q35-36 (nine families) and LQT3 on 3p21-24 (years) (Jiang et al., 1994). Half a dozen remain unconnected to known positions, indicating different position heterogeneity for LQT. This degree of heterogeneity suggests that separate LQT genes can encode proteins that interact to regulate cardiac repolarization and arrhythmia risk.

Little is known about the physiological function of LQT, but this disease is associated with the prolongation of the QT interval on the electrocardiogram, a signal of abnormal cardiac repolarization. This association suggests that the gene encoding the ion channel, or a regulatory factor thereof, is a suitable candidate for LQT. HRAS located on chromosome 11p15.5 was excluded as a candidate for LQT1 by linkage analysis (private observation) based on direct DNA sequence analysis (Roy et al., 1994). Neuroendocrine calcium channel genes (CACNL1A2; Chin et al., 1991; Seino et al., 1992) and genes encoding GTP binding proteins that regulate potassium channels (GNAI2; Weinstein et al., 1988; Magovcevic et al., 1992) was a candidate for LQT3 based on its chromosomal location. However, subsequent linkage analysis did not take this gene into account (Wang and Keating, private data). Skeletal muscle chloride channel (CLCN1; Koch et al., 1992) and cardiac muscarinic-acetylcholine receptor (CHRM2; Bonner et al., 1987) became candidates for LQT2 based on its chromosome 7q35-36 position, but later Linkage analysis excluded this gene (Wang et al., Reported).

In theory, mutations in the cardiac sodium channel gene can result in LQT. Voltage gated controlled sodium channels regulate rapid depolarization in ventricular myocytes and also conduct small currents during the stable phase of action potential (Attwell et al., 1979). Minor abnormalities in sodium channel function (eg, delayed sodium channel inactivation or altered voltage dependence of channel inactivation) can delay cardiac repolarization, leading to QT prolongation and arrhythmia. In 1992, Gellens and his colleagues cloned and characterized the cardiac sodium channel gene, SCN5A (Gellens et al., 1992). The structure of this gene was similar to other already characterized sodium channels, encoding the giant protein of the 2016 amino acid. This channel protein contains four homology domains (DI-DIV), each containing six putative membrane spanning segments (S1-S6). SCN5A has recently been mapped to chromosome 3p21 and has become an excellent candidate gene for LQT3 (George et al., 1995), which has since been found to be related to LQT3 (Wang et al., 1995a).

In 1994, Warmke and Ganetzky identified new human cDNA, human ether a-go-go related genes (HERG, Warmke and Ganetzky, 1994). HERG is localized on human chromosome 7 by PCR analysis of a somatic hybrid panel (Warmke and Ganetzky, 1994) and is a candidate for LQT2. The function of the protein encoded by HERG is unknown, but it has a predicted amino acid sequence homologous to the potassium channel. HERGs were isolated from hippocampal cDNA libraries by homology to the Drosophila ether a-go-go gene (eag), which encodes a calcium regulated potassium channel (Bruggeman et al., 1993). HERG is not a human homologue of eag, but has only -50% amino acid sequence homology. HERG has been shown to be associated with LQT2 (Curran et al., 1995).

A new potassium channel gene called KVLQT1 was discovered. Evidence is presented herein to indicate that KVLQT1 is LQT1. Sixteen families with mutations in KVLQT1 were identified and characterized and there was a complete binding between LQT1 and KVLQT1 in all 16 families. KVLQT1 maps to chromosome 11p15.5 and is a candidate gene for LQT1. KVLQT1 encodes a protein with the structural features of potassium channels, and expression of genes measured by Northern blot analysis demonstrated that KVLQT1 is the strongest expression in the heart. One intragenic deletion and ten other missense mutations causing LQT were identified in KVLQT1. These data define KVLQT1 as a new cardiac potassium channel gene and indicate that mutations in this gene cause sensitivity to ventricular tachyarrhythmias and sudden death.

I_Ks One component of the channel is known to be minK, a 130 amino acid protein with a single putative transmembrane domain (Takumi et al., 1988; Goldstein and Miller, 1991; Hausdorff et al., 1991; Takumi et al., 1991; Busch et al., 1992; Wang and Goldstein, 1995; Wang et al., 1996). It seems that the size and structure of this protein makes it impossible to form functional channels with minK alone (Attali et al., 1993; Lesage et al., 1993). KVLQT1 and minK associate together to form heart I_Ks Evidence to form potassium channels has been presented. I_Ks Dysfunction is the cause of deep vein.

Summary of the Invention

The present invention demonstrates the molecular basic principles of long-term QT syndrome. More specifically, the present invention has identified that molecular variants of the KVLQT1 gene cause or are associated with the development of LQT. Genotyping results indicate that KVLQT1 is fully linked to LQT1 in 16 unrelated families. Analysis of the KVLQT1 gene will enable early diagnosis of subjects with LQT. This diagnostic method consists of analyzing the DNA sequence of the KVLQT1 gene of a subject and comparing it with the DNA sequence of a normal non-variable gene. In a second embodiment, the KVLQT1 gene of the subject is screened for mutations that cause LQT. By predicting LQT, doctors may be able to prevent the disease with medical therapies such as beta blockers.

It has also been demonstrated that KVLQT1 and minK proteins associate together to form cardiac I _Ks potassium channels. I _Ks dysfunction is the cause of deep vein. The fact that these two proteins associate together to form I _Ks channels is useful for developing screening assays for drugs that are useful for treating or preventing LQT1. By co-expressing both wild type and its mutant genes in cells such as oocytes, drugs that have an effect on the I _Ks channel can be screened. This fact is also useful for analysis of the minK gene for early diagnosis of subjects with LQT. This diagnostic method is performed as described above for KVLQT1.

1 is a schematic structure of a portion of the LQT family 1532. Diseased individuals are represented by circles (females) or squares (males) filled with blanks, unmorbid individuals by empty symbols, and objects with an unclear phenotype by stipple method. Genotypes for chromosome 11 markers are shown as haplotypes just below each sign. The marker order (top to bottom) is Tel-HRAS-D11S922-TH-D11S1318-D11S454-D11S860-D11S12-Cen. The accuracy of the haplotype was ensured using genotypes from additional chromosome 11p15.5 markers (Q. Wang, closed results). Inferred genotypes are shown in parentheses. Disease chromosomes are indicated by boxes and recombination is indicated by dense horizontal lines. Diseased chromosomal recombination occurs in individuals: IV-22, IV-25, V-6, V-17, V-24, V-34, VI-13, VI-14 and VI-16. Recombination that occurs on non- diseased chromosomes is not shown. KVLQT1 is an SSCP isoform in KVLQT1 identified by primers 5 and 6, which is unique in K1532 and represents a disease related mutation (allele 2 is a mutant allele). Haplotype analysis shows that KVLQT1 is located between flanking markers D11S922 and D11S454.

2 is a physical map of the LQT1 region. The ideogram of chromosome 11 indicates the approximate location of LQT1 (11p15.5). The location of the polymorph markers and some cosmids are indicated by vertical lines on the map. In the precise genetic map, LQT1 is located between TH and D11S454. The distance between TH and D11S454 was evaluated as <700 kb by pulse meter gel analysis. The physical map of the minimum set of overlapping YAC and P1 clones is shown. The location of the KVLQT1 cDNA and the trapped exons are shown. The dashed lines of YACs represent chimeras.

3A and 3B show the nucleotide and inferred amino acid sequences of KVLQT1 (not including the region encoding the first 34 amino acids). (A) shows the complex sequence of KVLQT1. This nucleotide sequence is SEQ ID NO: 15. The amino acid sequence is SEQ ID NO: 16. Six estimated transmembrane segments S1 to S6 and estimated void area Pore are shown. Potential glycosylation sites (N160) are underlined. Two coincident polyadenylation signals are shown in bold in the 3 ′ untranslated region. Complex cDNA sequences for KVLQT1 were obtained by terminal sequencing of overlapping cDNA clones and also by primer walking. The KVLQT1 sequence was assigned GenBank Accession Number U40990. (B) shows the arrangement of the S1-S6 region of KVLQT1 with the Drosophila Shaker potassium channel, DMSHAKE1 (SHA) (Pongs et al., 1988). Sameness (|) and similarity (:) are indicated. The three separate fragments of KVLQT1 are SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, in that order. The three separate fragments of DMSHAKE1 are SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, in that order.

4 is a KVLQT1 tissue expression pattern. Northern analysis revealed 3.2 kb KVLQT1 mRNA in human kidney, lung, placenta, and heart, the highest of which was in the heart.

5A-5D show KVLQT1 missense mutations isolated concurrently with LQT in families K1532 (FIG. 5A), K2605 (FIG. 5B), K1723 (FIG. 5C) and K1807 (FIG. 5D). The results of SSCP analysis by primer pair 5-6 (K1532), primer pair 9-10 (K1723, K1807), and primer pair 11-12 (K2605) are shown below each diagram. The SSCP isoforms (marked with ^* ) are separated from the LQTs in each household. For K1532, only 8 out of 217 individuals were shown, and the results of SSCP analysis for additional members of K1532 are shown in FIG. 1 (KVLQT1 allele 2). The aberrant SSCP isoforms that simultaneously separate with LQT in K161 and K162 are the same as the aberrants defined in K1807, so no results were shown for this pedigree. The results of DNA sequencing of the normal and abnormal heteroforms (right) are shown below each system.

6A-6G show LQTs in households K13216 (FIG. 6A), K1777 (FIG. 6B), K20925 (FIG. 6C), K2557 (FIG. 6D), K13119 (FIG. 6E), K20926 (FIG. 6F), and K15019 (FIG. 6G). Combined KVLQT1 gene deletion and missense mutations. The results of SSCP analysis by primer pair 1-2 (K13216, K2557, K13119, K15019), primer pair 7-8 (K1777, K20926) and primer pair 9-10 (K20925) are shown below each system. The aberrant SSCP isoforms that simultaneously separate LQTs in K2050, K163 and K164 are the same as the aberrant variants defined in K1723 and K1807, so no results were shown for this pedigree. The DNA sequencing results of the normal heteromorphs (left) and the abnormal heteroforms (right) are shown below each system. The sequence is shown on the antisense strand.

7 is a schematic diagram of the predicted phase of KVLQT1 protein and the location of KVLQT1 mutations.

8A and 8B show the structure of human and Xenopus KVLQT1 and tissue expression patterns of human KVLQT1. (A) is a comparison of human and some Xenopus KVLQT1 amino acid sequences. Vertical lines represent identical residues. The Xenopus amino acid sequence is SEQ ID NO: 23 and the human amino acid sequence is SEQ ID NO: 24. (B) shows a northern analysis showing expression of KVLQT1 in human heart, placenta, lung, kidney and pancreas.

9A-9E show that simultaneous expression of KVLQT1 and hminK in CHO cells induces a current that is approximately equal to cardiac I _Ks . (A) shows the KVLQT1 current recorded during the 1 second depolarization pulse for a membrane potential of -50 to +40 mV applied from a holding potential of -80 mV. (B) shows normalized isochronous activation curves for cells transfected with KVLQT1 (n = 6; 1 sec pulse) or KVLQT1 and hminK (n = 7; 7.5 sec pulse). CE) is the current recorded in 7.5 second pulses for -40, -20, -10, 0, +20 and +40 mV in cells transfected with hminK (C), KVLQT1 (D) or KVLQT1 and hminK (E) Indicates. Tail currents were measured at -70 mV at D and at -50 mV at C and E. At +40 mV, the amplitude of steady-state KVLQT1 current was 0.37 ± 0.14 nA (n = 6). In cells cotransfected with KVLQT1 and hminK, the time dependent current in 7.5 sec pulses for +40 mV was 1.62 ± 0.39 nA (n = 7).

10A-10C show the expression of KVLQT1 in Xenofus oocytes. (A) shows the current recorded in oocytes injected with 12.5 ng KVLQT1 cRNA. Pulses were applied in 10 Hz increments from -70 to +40 Hz. (B) The isochronous (1s) activation curve for KVLQT1 current is shown. V _½ was -14.0 ± 0.2 mV was slope factor was 11.2 ± 0.2 ㎷ (n = 9 ). (C) shows that the relationship of E _rev to log [K ⁺ ] _e fits as a linear function with a slope of 49.9 ± 0.4 mm 3 (n = 6-7 oocytes per point). Tail current was measured at 5 to 6 voltage after 1.6 seconds prepulse for +10 mA.

11A-11E show co-expression of KVLQT1 and hminK, suggesting the presence of KVLQT1 homologues in Xenough oocytes. Current was -40, -20, 0, +20 and +40 ㎷ in oocytes injected with 5.8 ng KVLQT1 (FIG. 11A), 1 ng hminK (FIG. 11B), or co-injected with both cRNAs (FIG. 11C). Was recorded. FIG. 11D shows the current-voltage relationship measured using 2 sec pulses for KVLQT1 and hminK, or 7.5 sec pulses for KVLQT1 and hminK (n = 20 cells for each condition). hminK cRNA 60 pg or non-implanted with 1 ng for cell, I _sK at +40 ㎷ was 2.11 ± 0.12 ㎂ and 2.20 ± 0.18 ㎂. 11E is injected with hminK (V½ = 2.4 ± 0.3 Hz; slope = 11.4 ± 0.3 Hz; n = 16) or co-injected with KVLQT1 and hminK cRNA (V½ = 6.2 ± 0.3 Hz; slope = 12.3 ± 0.2 mm 3; n = 20) normalized isochronous activation curve for oocytes.

12A-12D show the nucleotide sequence for KVLQT1 cDNA and its translation product.

The present invention is directed to identifying whether LQTs are mapped to the KVLQT1 gene and that molecular variants of this gene cause LQT or are involved in the development of LQT. The invention also relates to confirming that KVLQT1 and minK are co-associated to form a cardiac I _Ks potassium channel. More specifically, the present invention relates to mutations in the KVLQT1 gene and its use in the diagnosis of LQT. The invention also relates to methods for screening humans for the presence of KVLQT1 gene variants that cause LQT. Since LQT can now be detected more clearly early (ie, before symptoms appear), better treatment choices will be made in individuals identified as having LQT. The present invention also relates to methods of screening drugs useful for treating or preventing LQT1.

The present invention provides a method of screening for the KVLQT1 gene to identify mutations. Such methods may also comprise amplifying a portion of the KVLQT1 gene and may also include providing a set of polynucleotides that are primers for amplifying said portion of the KVLQT1 gene. This method is useful for identifying mutations for use in diagnosing or predicting LQT.

Long-term QT syndrome is a hereditary disease that causes sudden death from deep veins, specifically tordes de pointes and ventricular fibrillation. LQT has already been mapped to three places: LQT1 on chromosome 11p15.5, LQT2 on 7q35-36 and LQT3 on 3p21-24. The inventors found that there is a genetic binding between LQT1 and the polymorph in KVLQT1, the cardiac potassium channel gene.

In addition, the present invention demonstrates that minK on chromosome 21 is also associated with LQT. minK protein and KVLQT1 associate together to form a K ⁺ channel. Accordingly, the present invention provides a method of screening for the minK gene to identify mutations. Such methods may also consist of amplifying a portion of the minK gene and may also comprise providing a set of polynucleotides that are primers for amplifying said portion of the minK gene. This method is useful for identifying mutations for use in diagnosing or predicting LQT.

Finally, the present invention relates to a method for screening drug candidates to identify drugs useful for treating or preventing LQT. Drug screening is performed by co-expressing mutant KVLQT1 and / or minK genes in cells such as oocytes, mammalian cells or transgenic animals and analyzing the effect of drug candidates on the I _Ks channel. This effect is compared with the I _Ks channel activity of wild type KVLQT1 and minK genes.

Evidence that the KVLQT1 gene is involved in causing LQT has been obtained by finding DNA sequences extracted from aberrant KVLQT1 gene products or affected family members forming abnormal gene products. Such LQT susceptible alleles will co-separate with large family diseases. They will be present more frequently in non-family individuals with LQT than in the general population. The key is to find mutations that are serious enough to cause obvious impediments to the normal functioning of gene products. Such mutations will take many forms. The most severe form would be to significantly alter the frame shift mutation or huge deletion or protein expression that causes the gene to encode the abnormal protein. Less severe disorder mutations are small in-frame deletions and ratios that will have a significant effect on the resulting protein, such as changes from or to cysteine residues, from basic amino acids to acidic amino acids, or vice versa, from hydrophobic amino acids to hydrophilic amino acids, and vice versa. Conservative base pair substitutions, or other mutations that will affect secondary or tertiary protein structure. Leading to silent mutations or conservative amino acid substitutions will generally be expected not to disrupt protein function.

In accordance with the diagnostic and predictive methods of the present invention, changes in the wild type KVLQT1 gene are detected. The method can also be performed by detecting wild type KVLQT1 genes and identifying the cause of LQT deficiency as a result of this locus. "Changes in genic genes" include all forms of mutations, including deletions, insertions, and point mutations in coding and noncoding regions. Deletion can occur only in the entire gene or in part of it. Point mutations can result from stop codons, frame shift mutations or amino acid substitutions. Celiac mutation occurs only in any tissue that is not genetic in the germ line. Germline mutations can be found in any tissue of the body and are genetic. Point mutation results can occur in regulatory regions, such as promoters of genes, leading to loss or reduction of mRNA expression. Point mutations may completely disrupt proper RNA processing, leading to a loss of KVLQT1 gene product expression or a decrease in mRNA stability or translational efficacy.

The presence of LQT can be confirmed by testing any tissue in humans for mutations in the KVLQT1 gene or minK gene. For convenience, the following description of the KVLQT1 gene will be given. However, the description is equally applicable to testing for minK gene mutations. For example, it will be demonstrated that LQT develops in people with the genetic germ line KVLQT1 mutation. This can be determined by testing DNA from any tissue of the human body. More simply, blood is taken and DNA is extracted from the blood cells. Fetal examination can also be done by testing fetal cells, placental cells or amniotic cells for mutations in the KVLQT1 gene. For example, changes in wild-type KVLQT1 alleles, whether by point mutations or by deletion, can be detected by any means discussed in the specification.

There are several methods that can be used to detect DNA sequence changes. Direct DNA sequencing, passive sequencing, or automated sequencing can detect sequence changes. Another method is single stranded polymorphism analysis (SSCP) (Orita et al., 1989). This method cannot detect all sequence changes. In particular, it may be optimal for detecting most DNA sequence changes, even when the DNA fragment size is larger than 200 bp. Although reduced detection sensitivity is a disadvantage, the increased throughput possible by SSCP makes it possible to use direct sequencing for the detection of mutations based on the study as a viable alternative of interest. Thereafter, fragments with transferred mobility to the SSCP gel are sequenced to confirm the exact nature of the DNA sequence change. Another method based on the detection of mismatches between two complementary DNA strands is clamped denaturing gel electrophoresis (CDGE) (Sheffield et al., 1991), heteroduplex analysis (HA) (White et). al., 1992) and chemical mismatch cleavage (CMC) (Grompe et al., 1989). None of the above methods will detect large deletions, duplications or insertions, and will not detect regulatory mutations that will affect the transcription or translation of the protein. Other methods that can detect this class of mutations, such as protein cleavage analysis or asymmetry analysis, detect only certain types of mutations and will not detect missense mutations. Common methods for detecting DNA sequence changes can be found in a recent report by Grompe (1993). Once the mutation is known, allele specific detection methods, such as allele specific oligonucleotide (ASO) hybridization, can be used to quickly screen multiple different samples for the same mutation.

Rapid preliminary analysis to detect polymorphism of DNA sequences can be done by observing DNA cleavage by one or more restriction enzymes, preferably by multiple restriction enzymes, in a series of Southern blots. Each blot contains a series of normal individuals and LQT cases. Southern blots showing hybridization fragments (different in length from the control DNA when probed to or near the KVLQTl site) indicate possible mutations. If restriction enzymes are used that produce very large restriction fragments, pulsed gel electrophoresis (PFGE) is used.

Detection of point mutations can be accomplished by molecular cloning of the KVLQT1 allele and sequencing of the allele using techniques known in the art.

There are six known methods for more complete, still indirect testing to identify the presence of sensitive alleles. 1) single strand morphology analysis (SSCP) (Orita et al., 1989); 2) modified gradient gel electrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989); 3) RNase protection assays (Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele specific oligonucleotides (ASOs) (Conner et al., 1983); 5) Lee. The use of proteins that recognize nucleotide mismatches, such as the collie mutS protein (Modrich, 1991); And 6) allele specific PCR (Rano and Kidd, 1989). For allele specific PCR, primers are used that hybridize at their 3 'end to a particular KVLQT1 mutation. If no special mutation is present, no amplification product is observed. An Application Repository Mutation System (ARMS) may be used as described in European Patent Application Publication No. 0332435 and Newton et al. (1989). Insertion and deletion of genes may also be detected by cloning, sequencing and amplification. In addition, restriction fragment length polymorphism (RFLP) probes for genes or surrounding marker genes can be used to record changes in alleles or insertions into polymorphic fragments. Such methods are particularly useful for screening affected individuals for the presence of mutations found in those individuals. Other techniques for detecting insertions and deletions as known in the art can be used.

In the first three methods (SSCP, DGGE and RNase protection assays), new electrophoretic bands appear. SSCP detects bands that differentiate and migrate because sequence changes cause differences in single-stranded, intramolecular base pairs. RNase protection involves digesting a mutant polynucleotide into two or more smaller fragments. DGGE uses a denaturing gradient gel to detect differences in the rate of migration of mutant sequences compared to wild type sequences. In allele specific oligonucleotide analysis, oligonucleotides are detected that detect specific sequences, and the analysis is performed by detecting the presence or absence of hybridization signals. In mutS analysis, the protein binds only to sequences that contain nucleotide mismatches of heterologous duplexes between mutant and wild type sequences.

Mismatches in accordance with the present invention are hybridized nucleic acid duplexes wherein the two strands are not 100% complementary. The lack of overall homology may be due to deletion, insertion, inversion or substitution. Mismatch detection can be used to detect point mutations in a gene or its mRNA product. Although this technique is less sensitive than sequencing, it is simpler to implement on a large number of samples. An example of mismatch degradation techniques is the RNase protection method. In the practice of the present invention, this method involves the use of labeled riboprobes complementary to the human wild type KVLQT1 gene coding sequence. MRNA or DNA and riboprobes isolated from humans are annealed (hybridized) together and then digested with the enzyme RNase A, which can detect some mismatches of the duplex RNA structure. If a mismatch is detected by RNase A, it degrades at the site of the mismatch. Thus, when annealed RNA preparations were separated on an electrophoretic gel matrix, if a mismatch was detected and resolved by RNase A, the RNA product would be observed to be smaller than the full length duplex RNA for riboprobe and mRNA or DNA. will be. Riboprobes need not be the full length of the mRNA or gene, but may be either segment. If riboprobes consist solely of mRNA or segments of genes, it will be necessary to use these multiple probes to screen the entire mRNA sequence for mismatch.

In a similar manner, DNA probes can be used to detect mismatches via enzymatic or chemical degradation (Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986). Mismatches can also be detected by mobility shifts in electrophoresis of mismatched duplexes relative to matched duplexes (eg, Cariello, 1988). Riboprobes or DNA probes can be used to amplify cellular mRNA or DNA that may contain mutations using PCR (see below) prior to hybridization. DNA changes in the KVLQT1 gene can also be detected using Southern hybridization, especially if the change is a total rearrangement such as deletion and insertion.

The DNA sequence of the KVLQT1 gene, which was amplified using PCR, can also be screened using an allele specific probe. Such probes are nucleic acid oligomers, each of which comprises a region of gene sequence with a known mutation. For example, one oligomer may be about 30 nucleotides in length corresponding to a portion of the gene sequence. Devices of such allele specific probes can be used to screen PCR amplification products to confirm the presence of mutations already identified in the gene. Hybridization of the amplified KVLQT1 sequence and allele specific probes can be done, for example, on nylon filters. Hybridization of specific probes under stringent hybridization conditions indicates the presence of the same tissue mutations as in allele specific probes.

The most obvious test for mutations at candidate positions is to directly compare genomic KVLQT1 sequences obtained from the control population and the patient. Further, for example, by sequencing messenger RNA after amplification by PCR, there is no need to confirm the exon structure of the candidate gene.

Mutations in patients outside the coding region of KVLQT1 can be detected by testing noncoding regions such as introns and regulatory sequences within or within a gene. Early determination that mutations in non-coding regions are important can be made by Northern blot experiments revealing abnormally sized or abundant messenger RNA molecules in the patient compared to the control.

Changes in KVLQT1 mRNA expression can be detected by any technique known in the art. Known methods include northern blot analysis, PCR amplification and RNase protection. Reduced mRNA expression indicates a change in wild type genes. Changes in wild-type genes can also be detected by screening for changes in wild-type KVLQT1 protein. For example, monoclonal antibodies immunoreactive with KVLQT1 can be used to screen tissue. Lack of family antigens will indicate mutations. Antibodies specific for the product of a mutant allele may also be used to detect mutant gene products. Such immunological assays can be performed in a convenient format known in the art. Examples include Western blots, immunohistochemical assays and ELISA assays. Any means of detecting altered KVLQT1 protein can be used to detect changes in wild type KVLQT1 gene. Functional assays such as protein binding confirmation can be used. In addition, assays that detect KVLQT1 biochemical function can be used. The discovery of the mutant KVLQT1 gene product indicates a change in the wild type KVLQT1 gene.

The mutant KVLQT1 gene or gene product may be detected in other human samples such as serum, feces, urine and sputum. The same techniques discussed above for the detection of mutant genes or gene products in tissues may be applied to other body samples. By screening such body samples, a simple early diagnosis of LQT can be made.

Primer pairs of the invention are useful for determining the nucleotide sequence of a particular KVLQT1 or minK using PCR. Single stranded DNA primer pairs for KVLQT1 can be annealed to sequences within or surrounding the KVLQT1 gene on chromosome 11 to prepare for amplification of DNA synthesis of the KVLQT1 gene itself. The single stranded DNA primer pair for minK can be annealed to the sequence within or surrounding the minK gene on chromosome 21 to prepare for amplification of DNA synthesis of the minK gene itself. This complete set of primers allows the synthesis of the nucleotide coding sequence of all genes, namely exons. This set of primers preferably allows the synthesis of both intron and exon sequences. Allele specific primers may be used. Such primers will only amplify the product in the presence of the mutant allele as a template by annealing only to the particular KVLQT1 mutant allele.

To facilitate subsequent cloning of the amplified sequence, the primer may have a restriction enzyme site sequence attached to its 5 'end. Thus, all of the nucleotides of the primers are derived from the KVLQT1 sequence or the sequence adjacent to KVLQT1, with the exception of some nucleotides needed to form restriction enzyme sites. Such enzymes and sites are known in the art. The primer itself can also be synthesized using techniques known in the art. In general, primers can be prepared using commercially available oligonucleotide synthesizers. Given the sequence of KVLQT1, the design of particular primers will be apparent to those skilled in the art.

Nucleic acid probes provided by the present invention are useful for several purposes. It can also be used for RNase protection for detecting the above point mutations and also for Southern hybridization to genomic DNA. Probes can be used to detect PCR amplification products. It may be used to detect mismatches with the KVLQT1 gene or mRNA using other techniques.

Individuals carrying the wild type KVLQT1 gene did not have LQT. However, mutations that inhibit the function of the KVLQT1 gene product are associated with the development of LQT. Thus, the presence of altered (or mutated) KVLQT1 genes that produce loss of function, or proteins with altered function, directly lead to LQT, which increases the risk of deep vein. To detect KVLQT1 gene mutations, biological samples are prepared and analyzed for differences between the sequence of the allele to be analyzed and the sequence of the wild type allele. Mutant KVLQT1 alleles can be identified initially by any of the techniques described above. Subsequently, the mutant allele is sequenced to identify the specific mutation of the particular mutant allele. Mutant alleles can also be identified initially by identifying mutant (modified) proteins using conventional techniques. Mutant alleles are then sequenced to identify specific mutations for each allele. Inducing mutations, particularly the altered function of proteins, is used in the diagnostic and predictive methods of the present invention.

It has also been found that KVLQT1 protein associates with minK protein. Thus, minK mutations that inhibit the function of the minK gene product are associated with the development of LQT. Thus, the presence of altered (or mutated) minK genes that produce loss of function, or proteins with altered function, directly lead to LQT which increases the risk of deep vein. To detect minK gene mutations, biological samples are prepared and analyzed for differences between the sequence of the allele to be analyzed and the sequence of the wild type allele. Mutant minK alleles can be identified initially by any of the techniques described above. Thereafter, the mutant alleles are sequenced to identify specific mutations of the mutated (changed) protein using conventional techniques. Mutant alleles are then sequenced to identify specific mutations for each allele. Subsequently, inducing mutations, especially the altered function of the protein, is used in the diagnostic and predictive methods of the present invention.

Justice

The present invention uses the following definitions.

"Probe". Polynucleotide polymorphs associated with KVLQT1 alleles susceptible to LQT can be detected by hybridization with polynucleotide probes that form stable hybrids with that of the target sequence under moderately stringent hybridization and wash conditions. Stringent conditions will be used if the probe is expected to be completely complementary to the target sequence. If some mismatch is expected, the stringency of hybridization can be relaxed, for example if a variant is expected that results in the probe not being completely complementary. Conditions are chosen that interfere with nonspecific / incidental binding, i.e. minimize noise. Such indications identify neutral DNA polymorphs as well as mutations and require further analysis to demonstrate detection of KVLQT1 sensitive alleles.

Probes for the KVLQT1 allele may be derived from the sequence of the KVLQT1 region or cDNA thereof. The probe may be of a suitable length that affects all or part of the KVLQT1 region and also enables specific hybridization to that region. If the target sequence contains the same sequence as the probe, the probe may be short in the range of about 8 to 30 base pairs, since the hybrid will be relatively stable under stringent conditions. If some mismatch is expected by the probe, ie if the probe is detected to hybridize to the variant region, a longer probe that hybridizes to the target sequence with the required specificity can be used.

The probe will comprise an isolated polynucleotide attached to a label or reporter molecule and can be used to separate other polynucleotide sequences having sequence similarity by standard methods. Techniques for preparing and labeling probes may be found, for example, in Sambrook et al., 1989 or Ausubel et al., 1992. Other similar polynucleotides may be selected by homologous polynucleotides. In addition, polynucleotides encoding such or similar polypeptides can be synthesized or selected using the redundancy of the genetic code. For example, various codon substitutions can be introduced by silent changes (by forming various restriction sites) or to optimize expression for a particular system. Mutations can be introduced to modify the properties of a polypeptide, perhaps to alter polypeptide degradation or conversion.

Probes consisting of synthetic oligonucleotides or other polynucleotides of the invention may be derived from chemical or synthetic or natural single or double stranded polynucleotides. Probes may be labeled by nick read, Klenow fill-in reactions or by other known methods.

Part of the polynucleotide sequence of less than about 6 kb, generally less than about 1.0 kb, having about 8 or more, generally about 15 nucleotides, obtained from the polynucleotide sequence encoding KVLQT1, is preferred as the probe. This probe can also be used to determine whether mRNA encoding KVLQT1 is present in a cell or tissue.

A "regulatory sequence" normally relates to a sequence within 100 kb of the coding region of a locus, but moreover coding regions and more, which affect gene expression (including transcription of genes and translation, splicing, stability, etc. of messenger RNA). It may be remote.

"Substantial homology or similarity". When the nucleic acid or fragment thereof is optimally aligned with another nucleic acid (or its complementary strand) (with the appropriate nucleotide insertion or deletion), at least about 60% of the nucleotide base, generally at least about 70%, more generally If at least about 80%, preferably at least about 90%, more preferably about 95-98% have nucleotide sequence identity, the nucleic acid or fragment thereof is "substantially homologous" ("or substantially similar") to another will be.

In addition, there is substantial homology or (similarity) when a nucleic acid or fragment thereof hybridizes to another nucleic acid (or its complementary strand), to one strand, or to its complement under selective hybridization conditions. Selectivity of hybridization exists when hybridization occurs that is substantially more selective than the lack of overall specificity. Typically, selective hybridization when at least about 55% homology, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90% homology to at least about 14 nucleotide stretches Will happen (see Kanehisa, 1984). Homologous length comparisons, which may be stretched longer, are in some embodiments often about 9 or more nucleotides, generally about 20 or more nucleotides, more generally about 24 or more nucleotides, typically about 28 or more nucleotides, more typically about 32 Abnormal nucleotides, preferably about 36 or more nucleotide stretches.

Nucleic acid hybridization is readily understood by those skilled in the art, in addition to base composition, length of complementary strands, and number of nucleotide base mismatches between hybridizing nucleic acids, depending on conditions such as salt concentration, temperature, or organic solvent. Will be affected. Stringent temperature conditions generally include a temperature of at least 30 ° C, typically at least 37 ° C, preferably at least 45 ° C. Stringent salt concentration conditions will typically be less than 1000 mM, typically less than 500 mM, preferably less than 200 mM. However, the combination of parameters is even more important than the measurement of any single parameter (eg, Wetmur & Davidson, 1968).

Probe sequences may be specifically hybridized to duplex DNA under any condition that forms a triplex or other high dimensional DNA complex. Preparation of such probes and appropriate hybridization conditions are known in the art.

Recombinant or chemically synthesized nucleic acids; Preparation of Vectors, Transformants, and Host Cells

Large amounts of polynucleotides of the invention can be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments for encoding the desired fragments will be incorporated into recombinant polynucleotide constructs, generally DNA constructs that can be introduced into and replicate in prokaryotic or eukaryotic cells. Generally, polynucleotide constructs will be suitable for replication in single cell hosts such as yeast or bacteria, but for introduction into cultured mammalian or plant or other eukaryotic cell lines (with and without assimilation into the genome). It may be. Purification of nucleic acids produced by the methods of the invention is described in Sambrook et al., 1989 or Ausubel et al., 1992.

The polynucleotides of the present invention may be produced by chemical synthesis, for example by the phosphoramidite method described in the literature or the triester method according to the literature, and may be carried out on a commercial, automated oligonucleotide synthesizer. Double stranded fragments can be obtained from single stranded products of chemical synthesis by synthesizing the complementary strands and annealing the strands together under appropriate conditions or by adding complementary strands using DNA polymerases with appropriate primer sequences.

Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may consist of a replication system recognized by the host, including a polynucleotide fragment encoding a given polypeptide, and is preferably operable to the polypeptide coding segment. And transcription and translation initiation control sequences that are well linked. Expression vectors include, for example, replication or autonomous replication sequences (ARS) and expression control sequences, promoters, enhancers and necessary processing information sites such as ribosomal binding sites, RNA splice sites, polyadenylation sites, transcription termination sequences. And mRNA stabilizing sequences. Such vectors can be prepared by standard recombinant techniques known in the art and are discussed, for example, in Sambrook et al., 1989 or Ausubel et al., 1992.

Appropriate promoters and other necessary vector sequences will be selected to be functional in the host, and may optionally include those naturally combined with the KVLQT1 or minK gene. Examples of viable combinations of cell lines and expression vectors are described in Sambrook et al., 1989 or Ausubel et al., 1992; Metzger et al., 1988. Many useful vectors are known in the art and can be purchased from vendors such as Stratagene, New England Biolabs, Promega Biotech, and the like. Promoters such as trp, lac and phage promoters, tRNA promoters and glycolysis promoters can be used in prokaryotic hosts. Useful yeast promoters include metallothionein, 3-phosphoglycerate kinase or other glycolysis enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, maltose and galactose And promoter regions for the like. Suitable vectors and promoters for use in yeast expression are described in more detail in Hitzeman, EP 73,675. Suitable non-natural mammalian promoters are derived from early and late promoters from SV40 (Fiers et al., 1978) or mouse molony leukemia virus, mouse tumor virus, avian sarcoma virus, adenovirus II, bovine papilloma virus or polyoma. Promoter can be mentioned. In addition, the construct may be linked to an amplifiable gene (eg, DHFR) such that multiple copies of the gene can be made. Suitable enhancers and other expression control sequences are described in Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, Cold Spring Harbor, New York (1983).

Such expression vectors can autonomously replicate but can also be replicated by methods known in the art by insertion into the genome of a host cell.

Expression and cloning vectors may comprise a selectable marker, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of these genes ensures the growth of only the host cell expressing the insert. Typical selection genes include a) conferring resistance to antibiotics or other toxic substances, such as ampicillin, neomycin, methotrexate, etc., b) supplementing supplemental nutritional deficiencies, or c) decisive nutrients that are not available in complex media, For example, it encodes a protein that supplies a gene encoding a D-alanine racease for Bacillus. The selection of appropriate selectable markers will depend on the host cell, and suitable markers for other hosts are known in the art.

The vector containing the nucleic acid is transcribed in vitro and the RNA formed is introduced into the host cell by methods known in the art, for example by injection (Kubo et al., 1988) or the vector is a host cell type. Methods known in the art, for example, electroporation, which vary according to; Transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other materials; Microprojectile impacts; Lipofection; Infection (if the vector is a pathogen such as the retroviral genome); And by other methods directly into the host cell (Sambrook et al., 1989 and Ausubel et al., 1992). In particular, the introduction of a polynucleotide into a host cell by any method known in the art, including the above, will be referred to herein as "transformation". Cells into which such nucleic acids are introduced are meant to include descendants of such cells.

Large amounts of nucleic acids and polypeptides of the invention can be prepared by expressing KVLQT1 or minK nucleic acids or portions thereof in a vector or other expression vehicle in an affinity prokaryotic or eukaryotic host cell. The most commonly used prokaryotic host is a strain of Escherichia coli, and other prokaryotes such as Bacillus subtilis or Pseudomonas can also be used.

Mammalian or other eukaryotic host cells, such as yeast, fibrous fungi, plants, insects, or amphibian or avian species, may be useful for the production of the proteins of the invention. Methods of proliferation of mammalian cells in culture are known per se (Jakoby and Pastan (eds.) 1979). Examples of commonly used mammalian host cell lines include VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, but for example high expression, certain glycosylation patterns, or other features. It will be understood by the skilled practitioner that other cell lines may be appropriate to provide.

Clones are selected using markers according to the vector structure scheme. The markers may be on the same or different DNA molecules, preferably on the same DNA molecule. In prokaryotic hosts, transformants can be selected for example by resistance to ampicillin, tetracycline or other antibiotics. The production of particular products based on temperature sensitivity may serve as appropriate markers.

Prokaryotic or eukaryotic cells transformed with the polynucleotides of the invention will be useful not only for the production of nucleic acids and polypeptides of the invention, but also for studying the characteristics of KVLQT1 or minK polypeptides.

Probes and primers based on the KVLQT1 gene sequence described herein are used to identify homologous KVLQT1 gene sequences and proteins in other species. Such gene sequences and proteins are used in the above diagnostic / prediction, treatment and drug screening methods for the species from which it was isolated.

How to use: drug screening

The invention is particularly useful for screening compounds using KVLQT1 and minK proteins of transformed cells, transfected oocytes or transgenic animals. Since mutations in the KVLQT1 or minK protein may alter the function of the cardiac I _Ks potassium channel, cells with normal KVLQT1 or minK protein and mutant minK or KVLQT1 proteins, respectively, or mutant KVLQT1 and mutant minK proteins, are used for the channel. Candidate drugs are screened for effects. The drug is added to cells in culture or administered to transgenic animals and the effect on the induced current of I _Ks potassium channel is compared with the induced current of cells or animals containing wild type KVLQT1 and minK. Drug candidates that change the induced current to more normal concentrations are useful for treating or preventing LQT.

How to use: nucleic acid diagnostics and diagnostic kit

In order to detect the presence of KVLQT1 or minK alleles that make individuals susceptible to LQT, biological samples such as blood are prepared and analyzed for the presence or absence of sensitive alleles of KVLQT1 or minK. To detect the presence of LQT or as a predictive indicator, biological samples are prepared and analyzed for the presence or absence of mutant alleles of KVLQT1 or minK. Such test results and interpretation information are passed to the healthcare provider for contact with the subject. Such diagnosis is performed by a diagnostic laboratory or, alternatively, a diagnostic kit is prepared and sold to a healthcare provider or to an individual for self-diagnosis.

Initially, screening methods involve amplification of relevant KVLQT1 or minK sequences. In another preferred embodiment of the invention, the screening method comprises a non-PCR based method. Such screening methods include two-step label amplification methods known in the art. Both PCR and non-PCR based screening methods can detect the target sequence with high sensitivity.

The most preferred method currently used is target amplification. Here, the target nucleic acid sequence is amplified with a polymerase. One particularly preferred method of using polymerase induced amplification is polymerase chain reaction (PCR). Polymerase chain reactions and other polymerase induced amplification assays can achieve a million-fold increase in copy number using polymerase induced amplification cycles. Once amplified, the nucleic acid formed can be sequenced or used as a substrate for DNA probes.

When the probe is used to detect the presence of the target sequence, the biological sample to be analyzed, such as blood or serum, can be processed to extract nucleic acid as needed. Sample nucleic acids can be prepared by a variety of methods, such as denaturation, restriction digestion, electrophoresis or dot blotting, to facilitate detection of the target sequence. The targeted region of the assay nucleic acid generally must be at least partially single stranded to form a hybrid with the target sequence of the probe. If the sequence is naturally single stranded, denaturation will not be necessary. However, if the sequence is double stranded, the sequence will need to be denatured. Denaturation can be performed by various techniques known in the art.

Analytical nucleic acids and probes are cultured under conditions that promote stable hybrid formation of the putative targeted sequence of the analyte with the target sequence within the probe. The region of the probe used to bind the analyte can be made completely complementary to the target region of human chromosome 11 relative to KVLQT1. Therefore, very stringent conditions are desired to prevent inaccurate positive reactions. However, very stringent conditions are only used if the probe is complementary to a region of the unique chromosome in the genome. The stringent conditions of hybridization are determined by many factors, such as temperature, ionic strength, base composition, probe length and formamide concentration, during hybridization and during washing procedures. Such factors are outlined, for example, in Manatis et al., 1982 and Sambrook et al., 1989. In some situations, formation of high dimensional hybrids such as triplex, quadraplex, etc. may be desirable to provide a means for detecting target sequences.

In any case, the measurement of the resulting hybrid is generally made by the use of labeled probes. Alternatively, the probe can be unlabeled, but can be detected directly or indirectly by specific binding to the labeled ligand. Suitable labels and probe and ligand labeling methods are known in the art and include, for example, radiolabels, biotin, fluorescent, chemical, which may be included by known methods (eg nick detoxification, random priming or kinase). Light emitters (eg, dioxetane, especially triggered dioxetane), enzymes, antibodies, and the like. Such basic tissue changes are known in the art and include those that facilitate the separation of hybrids to be detected from foreign material and / or amplify signals from labeled residues. Many such methods of change are described in Matthews & Kricka, 1988; Landegren et al., 1988; US Pat. No. 4,868,105 and European Patent Publication No. 225,807.

As noted above, non-PCR based screening assays are also included in the present invention. This procedure hybridizes nucleic acid probes (or analogs, such as the methylphosphonate backbone in place of normal phosphodiester) to low concentration DNA targets. The probe may have an enzyme covalently bound to the probe such that the covalent bond does not interfere with the specificity of hybridization. This enzyme-probe-conjugate-target nucleic acid complex can be separated from the free probe enzyme conjugate and the substrate is added for enzyme detection. Enzyme activity is observed as a change in chromogenic or luminescent product, with increased sensitivity of 10 ³ -10 ⁶ . For example, see Jalonski et al., 1986 for examples of preparing oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes.

Two-step label amplification methods are known in the art. Small ligands (eg, digoxigenin, biotin, etc.) are based on the principle of attachment to nucleic acid probes capable of specifically binding KVLQT1. Allele specific probes are predicted within the scope of this example and exemplary allele specific probes include probes that achieve prone mutations in this patent application.

In one embodiment, small ligands attached to nucleic acid probes are specifically recognized by antibody-enzyme conjugates. In one embodiment of this example, digoxigenin is attached to a nucleic acid probe. Hybridization is detected by antibody-alkali phosphatase conjugates that convert chemiluminescent substrates. For a method of labeling nucleic acid probes according to this embodiment, reference may be made to the literature (Martin et al., 1990). In a second embodiment, the small ligand is recognized by a second ligand-enzyme conjugate that can specifically complex to the first ligand. A well known embodiment of this example is the biotin-avidin type interaction. For methods of labeling nucleic acid probes and their use in biotin-avidin basic assays, see Rigby et al., 1977 and Nguyen et al., 1992.

It is anticipated within the scope of the present invention that the nucleic acid probe assay of the present invention will utilize a nucleic acid probe cocktail capable of detecting KVLQT1 or minK. Thus, in one example of detecting the presence of KVLQT1 in a cell sample, one or more probes complementary to the gene are used, in particular many other probes being two, three or five different nucleic acid probe sequences. In another embodiment, one or more probes complementary to this gene are used to detect the presence of a mutation in the patient's KVLQT1 gene sequence, wherein the cocktail will bind to allele specific mutations identified in the patient population where KVLQT1 has changed. Probes that may be present. In this embodiment, any number of probes may be used and preferably comprise probes corresponding to major gene mutations identified as individuals susceptible to LQT.

How To: Peptide Diagnostics and Diagnostic Kits

The presence of LQT can also be detected based on changes in wild type KVLQT1 or minK polypeptides. Such changes can be identified by sequence analysis according to conventional techniques. More preferably, the antibody (polyclonal or monoclonal) is used to detect the difference or absence of KVLQT1 or minK peptide. Techniques for forming and purifying antibodies are known in the art and any such technique can be selected to achieve the manufacturing methods claimed in the present invention. In a preferred embodiment of the invention, the antibody will not only immunoprecipitate KVLQT1 or minK protein from solution, but will also react with this protein on an immunoblot of a western or polyacrylamide gel. In another preferred embodiment, the antibody will detect KVLQT1 or minK in paraffin or frozen tissue fragments using immunocytochemical techniques.

Preferred embodiments of methods for detecting KVLQT1 or minK or mutations thereof include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoassay, including sandwich assays using monoclonal and / or polyclonal antibodies. Radioactive assay (IRMA) and immunoenzyme assay (IEMA). Exemplary sandwich analyzes are described in David et al., US Pat. Nos. 4,376,110 and 4,486,530, which are incorporated herein by reference.

How To Use: Gene Therapy

According to the present invention, a method is provided for supplying wild type KVLQT1 or minK function to cells with mutant KVLQT1 or minK alleles, respectively. Provision of such a function should allow for normal functionalization of the recipient cell. The wild type gene or part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from an extrachromosomal location. More preferred is the situation where the wild type gene or part thereof is introduced into the mutant cell to recombine with the endogenous mutant gene present in the mutant cell. Such recombination requires double recombination to allow for modification of the gene mutation. Vectors for introducing genes for recombinant and extrachromosomal maintenance are known in the art and any suitable vector can be used. Methods of introducing DNA into cells, such as electroporation, simultaneous precipitation of calcium phosphate and viral transduction, are known in the art and the choice of methods is within the authority of the practitioner.

In general, as discussed above, KVLQT1 or minK genes or fragments may be used in gene therapy to increase the amount of expression products of such genes in cells. It may be useful to increase the expression level of a given LQT gene even in cardiac cells in which mutant genes are expressed at "normal" levels, but such gene products are not fully functional.

Gene therapy will be performed according to generally accepted methods as described in Friedman, 1991. Cells obtained from the patient will be first analyzed by this diagnostic method to confirm the production of KVLQT1 or minK polypeptides in the cells. Virus or plasmid vectors (described in more detail below) are prepared that contain copies of the KVLQT1 or minK genes that are linked to expression regulatory elements and can replicate within the cell. Suitable vectors are known as described in US Pat. No. 5,252,479 and PCT Publication Application WO93 / 07282. Inject this vector into the patient. If the transfected gene is not permanently inserted into the genome of each targeted cell, it can be repeated periodically.

Gene delivery systems known in the art may be useful for the practice of the gene therapies of the invention. This includes viral and nonviral delivery methods. Numerous viruses have been used as gene transfer vectors, examples of which are papovaviruses (eg SV40, Madzak et al., 1992), adenoviruses (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vaccine virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese et al., 1990), and birds Retroviruses (Brandyopadhyay and Temin, 1984; Petropoulos et al., 1992), mice (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al., 1988) , And human sources (Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992). Most human gene therapy procedures are based on disabled mouse retroviruses.

Nonviral gene delivery methods known in the art include chemical techniques such as calcium phosphate co-precipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); Mechanical techniques such as microinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster et al., 1981; Constantini and Lacy, 1981); Membrane fusion mediated delivery via liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda et al., 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1992) ; And direct DNA uptake and receptor mediated DNA delivery (Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al., 1989; Wolff et al., 1991; Wagner et al. , 1990; Wagner et al., 1991; Cotten et al., 1990; Curiel et al., 1991a; Curiel et al., 1991b).

In a combination of biological and physical gene delivery methods, plasmid DNA of any size is combined with polylysine-conjugated antibodies specific for adenovirus hexon proteins, and the complexes formed are bound to adenovirus vectors. Trimole complexes are used to infect cells. Adenovirus vectors allow for efficient binding, transmigration, and degradation of endosomes before the coupled DNA is damaged.

The liposome / DNA complex has been shown to be a direct mediator of gene transfer in vivo. Although the gene transfer process in standard liposome preparation is nonspecific, localized in vivo uptake and expression has been reported in tumor attachments, for example in the following direct in situ administration (Nabel, 1992).

Gene delivery techniques that target DNA directly to heart tissue are preferred. Receptor mediated gene delivery is accomplished by conjugating DNA (usually in the form of covalently closed supercoil plasmids) to protein ligands via polylysine. Ligands are selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell / tissue type. Ligand-DNA conjugates can be injected directly into the blood when needed and directed to target tissues where receptor binding and inward fulfillment of the DNA-protein complex occur. To overcome the problem of intracellular destruction of DNA, co-infection with adenoviruses can be enclosed to disrupt endosomal function.

The therapy is as follows: Patients with KVLQT1 or minK sensitive alleles are treated with a gene delivery vehicle so that some or all of their cardiac precursor cells receive at least one additional copy of a functional normal KVLQT1 or minK allele. . At this stage, the treated subjects reduced the risk of LQT to such an extent that the effects of sensitive alleles were countered by the presence of normal alleles.

How to use: transformed host

Animals for testing therapeutic agents may be selected after mutation of the entire animal or after treatment of germline lineage cells or conjugates. Such treatment generally involves the insertion of mutated KVLQT1 and / or minK alleles as well as the disrupted homologous genes obtained from the second animal species. Alternatively, endogenous KVLQT1 or minK genes in animals can be disrupted by insertion or deletion mutations or other genetic changes using conventional techniques (Capecchi, 1989; Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992; Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992; Donehower et al., 1992). After administration of the test substance to the animal, the presence of LQT should be assessed. If the test substance prevents or inhibits the sign of LQT, the test substance is a candidate therapeutic for LQT treatment. This animal model provides an extremely important test vehicle for potential therapeutic products.

Two methods have been used herein to identify LQT genes, candidate gene approaches and positional cloning. The information on the current position is LQT1 mapped to chromosome 11p15.5 (Keating et al., 1991a; Keating et al., 1991b), LQT2 mapped to 7q35-36 and LQT3 mapped to 3p21-24 (Jiang et al., 1994 are available for three LQT positions. The present invention also identified minK on chromosome 21 as the LQT gene. Candidate genetic approaches rely on mechanistic hypotheses based on physiology. Little is known about the physiology of LQT, but this disease is associated with prolongation of the QT interval on the electrocardiogram, a signal of abnormal cardiac repolarization. This association suggests that the gene encoding the ion channel, or a regulatory factor thereof, is a suitable candidate for LQT. This hypothesis is supported by the discovery that chromosome 7-linked LQT now arises from mutations in HERG, the putative cardiac potassium channel gene. Neuroendocrine calcium channel genes (CACNL1A2; Chin et al., 1991; Seino et al., 1992) and genes encoding GTP-binding proteins that regulate potassium channels (GNAI2; Weinstein et al., 1988; Magovcevic et al , 1992) are candidates for LQT3 based on their chromosomal location. However, subsequent binding assays excluded these genes (Wang and Keating, private data). It has been found that LQT3 is related to SCN5A (Wang et al., 1995a). However, despite considerable effort, candidate genetic approaches to chromosome 11-linked LQT have not been successful. Two potassium channel genes (KCNA4 and KCNC1) were mapped to the short arm of chromosome 11 (Wymore et al., 1994), but both were excluded as candidates for LQT1 by binding assays (Russell et al., 1995; present; Research). All other cardiac potassium, chloride, sodium and calcium channel genes already characterized were likewise excluded on the basis of their chromosomal location. The study of the present invention used positional cloning and mutation analysis to identify LQT1.

The present invention used genotyping to indicate that KVLQT1 is closely linked to LQT1 in 16 unrelated families (the examples are detailed). KVLQT1 is a putative cardiac potassium channel gene and results in chromosome 11-linked forms of LQT. Genetic analysis suggests that KVLQT1 codes for voltage gate controlled potassium channels of functional importance in cardiac repolarization, and KVLQT1 now associates with minK to form cardiac I _Ks potassium channels. If correct, the mechanism of chromosome 11-linked LQT will probably include reduced repolarization KVLQT1 current. Since potassium channels with six transmembrane domains are thought to be formed from homo- or hetero-tetramers (MacKinnon, 1991; MacKinnon et al., 1993; Covarrubias et al., 1991), LQT related mutations in KVLQT1 It can work through dominant-negative mechanisms. The type and location of the KVLQT1 mutations described herein is consistent with this hypothesis. The resulting inhibition of potassium channel function will now lead to an increased risk of abnormal cardiac repolarization and ventricular fibrillation. The biophysics of the mutations and potassium channel alpha subunits identified in HERG suggest that chromosome 7-linked LQTs arise from dominant-negative mutations and the resulting decrease in functional channels. In chromosome 3-bound LQTs, in contrast, LQT-related deletions identified in SCN5A are prone to the formation of functional cardiac sodium channels with altered properties such as delayed inactivation or altered voltage-dependence of channel inactivation. Delayed sodium channel inactivation will increase the internal sodium current which depolarizes the membrane. This effect is similar to the membrane potential change expected from HERG mutations where the external potassium current is reduced. It will not be easy for more harmful mutations of SCN5A to cause LQT. Reduction of the total number of cardiac sodium channels is expected to reduce the duration of action potentials, which are, for example, the opposite phenotype of LQT.

Pre-diagnosis of LQT was dependent on the identification of QT prolongation on the ECG. Unfortunately, ECG was rarely performed in young, healthy individuals. In addition, many LQT gene carriers have relatively normal QT intervals, and the first signal of the disease may be fatal deep vein (Vincent et al., 1992). Currently a third LQT gene (KVLQT1) has been identified, minK is also associated with LQT, and genetic testing for this disease can be planned. This will require subsequent mutation analysis and identification of additional LQT genes. By more detailed phenotypic analysis, phenotypic differences between the variable forms of LQT can be found. Such differences can be useful for diagnosis and treatment.

The identification of the association between the SCN5A, HERG and KVLQT1 gene mutations and LQT allows for early prescreening screening of individuals to confirm the risk of developing LQT. To identify such individuals, the SCN5A, HERG and / or KVLQT1 alleles are screened for mutations either directly or after cloning the allele. minK mutations can be found as well. Alleles were tested for the presence of normal alleles and other nucleic acid sequences using any suitable technique, including but not limited to: fluorescence in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis Single strand morphology analysis (SSCP), binding assay, RNase protection assay, allele specific oligonucleotide (ASO), dot blot analysis and PCR-SSCP analysis. For example, (1) determine and compare the nucleotide sequence (coding sequence or genomic sequence) of the cloned allele and the normal KVLQT1 gene or appropriate fragment, or (2) the RNA transcript of the KVLQT1 gene or gene fragment from the subject. The resulting single stranded whole genomic DNA is hybridized and the formed heterologs are treated with ribonuclease A (RNase A) and run on denaturing gels to detect any mismatch positions. These two methods can be carried out according to the following procedure.

The allele of KVLQT1 or minK of the subject was cloned using conventional techniques. For example, a blood sample is taken from the subject. Genomic DNA isolated from cells in this blood is partially digested to an average fragment size of about 20 kb. Separate fragments ranging from 18-21 kb. The formed fragment is ligated to the appropriate vector. The clone's sequence is identified and compared with the normal KVLQT1 or minK gene.

Alternatively, polymerase chain reactions (PCRs) are performed with primer pairs for the 5 'region or exon of the KVLQT1 gene. PCRs can also be performed with primer pairs based on any sequence of the normal KVLQT1 gene. For example, primer pairs for one of the introns are prepared and used. Finally, PCR may be performed on mRNA. The amplified product is analyzed by single stranded polymorphism (SSCP) using conventional techniques to identify any differences and sequence them and compare with normal gene sequences.

Amplify the DNA of the individual with the appropriate primer pairs and analyze the amplified product by dot-blot hybridization using, for example, an allele specific oligonucleotide probe to quickly screen the individual for a conventional KVLQT1 or minK gene.

The second method uses RNase A to help detect differences between normal KVLQT1 or minK genes and defective genes. This comparison is performed step by step using small (˜500 bp) restriction fragments of the KVLQT1 or minK gene as probes. Initially, the KVLQT1 or minK gene is digested with restriction enzyme (s) that cleave the gene sequence into fragments of about 500 bp. This fragment is separated on an electrophoretic gel, purified from the gel and cloned into SP6 vectors (eg pSP64 or pSP65) separately in both directions. SP6 based plasmids containing inserts of KVLQT1 or minK gene fragments are generated using an SP6 transcription system known in the art to generate both stranded radiolabeled RNA transcripts of a gene in the presence of [α- ³² P] GTP. Transcribed in vitro.

Individually, these RNA transcripts are used to form heterologous alleles with conventional techniques. Degradation of RNA strands occurs when treated with RNase A as a result of mismatches that occur in RNA: DNA heterologies due to sequence differences between KVLQT1 or minK fragments and KVLQT1 or minK allele subclones obtained from an individual. Such mismatches may be the result of point mutations or small deletions of an allele of an individual. Degradation of the RNA strand forms two or more small RNA fragments, which run faster on the denaturing gel than the RNA probe itself.

Any difference found will identify individuals with molecular variants of the KVLQT1 or minK gene and with the resulting long-term QT syndrome. Such variants may take many forms. The most severe form would be to significantly alter frame shift mutations or large deletions or protein expression that cause coding for the aberrant protein in the gene. Less severe disorder mutations are small in-frame deletions and ratios that will have a significant effect on the resulting protein, such as changes from or to cysteine residues, from basic amino acids to acidic amino acids, or vice versa, from hydrophobic amino acids to hydrophilic amino acids, and vice versa. Conservative base pair substitutions, or other mutations that will affect secondary or tertiary protein structure. Leading to silent mutations or conservative amino acid substitutions will generally be expected not to disrupt protein function.

Doctors may be able to identify individuals for risk of LQT at birth or even before birth by genetic testing. Prediagnosis of LQT will enable the prevention of such diseases. Current medical therapies, including beta adrenergic blockers, can prevent and delay the onset of problems associated with the disease. Finally, the present invention changes the understanding and treatment of the causes of common heart disease, such as deep vein, which causes 11% of all natural deaths. Current diagnostics focus on measuring QT intervals from electrocardiograms. This method is not a completely accurate indicator of the presence of long-term QT syndrome. The present invention is a more accurate indicator of the presence of this disease. Genetic testing and improved mechanistic understanding of LQT provide an opportunity for the prevention of life-threatening arrhythmias through proper therapy. If possible, for example, potassium channel openers will reduce the risk of arrhythmia in patients with KVLQT1, minK or HERG mutations, whereas sodium channel blockers are more effective for patients with mutations that alter the function of SCN5A. You can treat it. Finally, these studies provide insight into the mechanisms underlying conventional arrhythmias, as conventional arrhythmias are often associated with abnormal cardiac repolarization and can arise from genetic and acquired complex factors.

The invention will be described in more detail in the following examples, which are provided for purposes of illustration only and should not be construed as limiting the invention in any sense. Standard techniques known in the art or those specifically described below are used.

Example 1

Phenotype evaluation method

For this study, six large LQT families (K1532, K1723, K2605, K1807, K161 and K162) and some small households and sporadic cases were studied. LQT patients were identified from clinicians throughout North America and Europe. Two factors were considered for the phenotype: 1) medical history data (fainting faint, frequency of fainting, sudden seizure, age of onset of symptoms, and sudden death); And 2) QT interval on the ECG corrected for heart rate (QT _c ) (Bazzett, 1920). To avoid misclassifying individuals, we used the same conservative approach as phenotypic designation that was successful in previous studies (Keating et al., 1991a; Keating et al., 1991b; Jiang et al., 1994). Informed consent was obtained from each individual or his guardian in accordance with local institutional review board guidelines. Phenotypic data were interpreted without knowledge of genotype. Subjects with signs with a corrected QT interval (QT _c ) of 0.45 seconds or more and those without signs with QT _c of 0.47 seconds or more were classified as affected. Subjects with no signs with a QT _c of 0.41 sec or less were also classified as not affected. Subjects with no signs with QT _c between 0.41 and 0.47 seconds and subjects with signs with QT _c less than 0.44 seconds were classified as unclear.

Example 2

Genotyping and Linkage Analysis

Genomic DNA was prepared from peripheral blood lymphocytes or cell lines derived from Epstein-Barr virus transformed lymphocytes using standard techniques (Anderson and Gusella, 1984). For genotyping, four small tandem repeat (STR) polymorphs pre-mapped to chromosome 11p15.5 were used: D11S922, TH, D11S1318 and D11S860 (Gyapay et al., 1984). Genotyping of the RFLP markers (HRAS1, D11S454 and D11S12) was performed as described in Keating et al., 1991a.

Paired linkage analysis was performed using MLINK in LINKAGE v5.1 (Lathrop et al., 1985). The hypothesis of permeability of 0.90 and LQT gene frequency of 0.001 was used. Gene frequencies were assumed to be the same for men and women. Male and female recombination frequencies were considered equal. Using n as the number of alleles observed, the STR allele frequency was 1 / n. Although the maximum LOD score for D11S454 was confirmed at recombination rate 0, the presence of one non-absolute recombination (individual VI-14, FIG. 1) forms the LQT gene telomer of D11S454.

Example 3

Physical mapping

Primers were designed based on sequences from the TH-INS-IGFII and D11S454 sites and clones were identified and isolated from CEPH YAC libraries using PCR basic techniques (Green and Olson, 1990; Kwiatowski et al., 1990). YAC terminal sequences were determined by reverse PCR as described in Ochman et al., 1988 and used as STSs.

Isolation of P1 clones using single copy probes from previously identified cosmid cosQW22 (this study), cCI11-469 (D11S679), cCI11-385 (D11S551), cCI11-565 (D11S601), cCI11-237 (D11S454) (Tanigami et al., 1992; Tokino et al., 1991; Sternberg, 1990). Newly isolated P1s were mapped to chromosome 11p15 by FISH or Southern analysis. Terminal specific riboprobes were generated from newly isolated P1s and used to identify additional contiguous clones (Riboprobe Gemini Core System Kit; Promega). DNA for P1 and cosmid clones were prepared using alkaline soluble plasmid separation and purified by equilibrium centrifugation with a CsCl-ethydium bromide gradient as described in Sambrook et al., 1989. P1 insertion terminal sequences were determined by cycle sequencing as described in Wang and Keating, 1994. STSs were generated based on this insertion terminal sequence. The overlap between P1s and cosmid was calculated by summing up the common restriction fragments.

Example 4

Isolation and Characterization of KVLQT1 Clone

Adult human cardiac cDNA libraries (Stratagene) were plated and 1 × 10 ⁶ plaques were screened using exon 4181A trapped as a probe. Oligonucleotide probes for cDNA library screening were designed using the sequence of trapped exon 4181A. 1 × 10 ¹¹ clones were screened from the human cardiac cDNA library (Life Technologies, Inc.) using the GENETRAPPER® cDNA Positive Selection System. The sequence of the capture and recovery oligonucleotide was 5'-CAGATCCTGAGGATGCT-3 '(SEQ ID NO: 1) 5'-GTACCTGGCTGAGAAGG-3' (SEQ ID NO: 2).

Complex cDNA sequences for KVLQT1 were obtained by terminal sequencing of duplicate cDNA clones and also by primer walking. Sequencing was carried out in Pharmacia A.L.F. Automatically using an automated sequencer or manually using a Sequencenase Version 2.0 DNA Sequencing Kit (United States Biochemical, Inc.). Database analysis and sequencing were performed using BLAST network service, GCG software package, and IG software package from National Center for Biotechnology Information.

The partial genomic structure of KVLQT1 (transmembrane domains S2 to S6) was confirmed by cycle sequencing of P1 18B12 as described in Wang and Keating, 1994. Primers were designed based on the KVLQT1 cDNA sequence and used for cycle sequencing.

Example 5

Mutation analysis

SSCP was performed as described in Wang et al., 1995a; Wang et al., 1995b. Normal and abnormal SSCP products were isolated and sequenced directly as described in Wang and Keating, 1994 or subcloned with pBluescript (SK +; Stratagene) using the T-vector method (Marchuk et al., 1990). When using the latter method, several clones were sequenced by dideoxy chain termination method using Sequenase® version 2.0 (United States Biochemicals, Inc.).

Example 6

Northern analysis

Multiple tissue northern filters (Human MTN blot 1, Clontech) were probed with ³² P-labeled KVLQT1 cDNA probes as described in Curran et al., 1995.

Example 7

Accurate genetic and physical alignment of LQT1

The exact location of LQT1 was determined by genotyping of family 1532 (K1532), a large family in Northern Utah (FIG. 1). This pedigree was first used in early studies linking the LQT gene, LQT1 to chromosome 11p15.5 (Keating et al., 1991a; Keating et al., 1991b). Other family members were identified and phenotyped for the total sample size of 217 subjects. Phenotypic determination was performed as described in Keating et al., 1991a; Keating et al., 1991b; Jiang et al., 1994. Preliminary genotyping with markers in HRAS, TH, D11S454 and D11S12 included all identified members of K1532. These experiments confirmed the family's information branch. Further genotyping was performed using three high polymorphic markers D11S922, D11S1318 and D11S860 from chromosome 11p15.5 (Gyapay et al, 1994). Genotype and paired LOD scores for each marker are shown in FIG. 1 and Table 1. Of these markers, TH and D11S1318 were fully linked. Recombination was confirmed with all other tested markers, including HRAS, but in each case a statistically significant positive LOD score (+3 or higher) was identified. This data indicates that LQT1 is fully linked to TH and D11S1318 in this family and that the disease gene is located in the centromer of HRAS.

For accurate localization of LQT1, haplotype analysis of K1532 was performed (see FIG. 1). Nine chromosomal retention information recombinations were identified. Telomeric recombination is performed on uninfected subjects IV-22 (between D11S922 and TH), affected subjects IV-25 (between D11S922 and TH), unaffected subjects V-6 (between HRAS and D11S922), and affected subjects V-. Observed at 24 (between HRAS and D11S922). Centromeric recombination involves uninfected subject V-17 (between D11S860 and D11S454), affected subject V-24 (between D11S860 and D11S454), uninfected subject V-34 (between D11S860 and D11S454), and uninfected subject VI. -13 (between D11S860 and D11S454), unaffected subject VI-14 (D11S454 and D11S1318) and affected subject VI-16 (between D11S860 and D11S454). With a recent study of the LQT1 centromer position of TH (Russell et al., 1995), this data relates to the position of LQT1 in the interval between TH and D11S454.

Pulsed gel analysis by genomic probe from chromosome 11P15.5 was used to assess the size of the region containing LQT1. Probes from TH, D11S551 and D11S454 hybridized to 700 kb Mlu I restriction fragments (FIG. 2). This data indicates that the region containing LQT1 is less than 700 kb. This region was physically labeled by screening a P1 library and yeast artificial chromosome (YAC) with probes from that region (Tanigami et al., 1992; Tokino et al., 1991). The order of this clone was confirmed using fluorescence in situ hybridization (FISH) analysis as follows: telomer-TH-D11S551-D11S679-D11S601-D11S454-centromer. Adjacent duplicate clones were identified using the clones identified in the initial experiment. The minimum set of clones from the LQT1 interval is shown in FIG. 2.

Paired LOD score between LQT1 and 11p15.5 markers Recombination Rate (θ) 0.0 0.001 0.01 0.05 0.1 0.2 Z _max ^* θ _max + HRAS 9.67 9.94 10.50 10.38 9.62 7.57 10.59 0.021 D11S922 10.05 13.05 13.85 13.59 12.59 10.01 13.92 0.019 TH 11.01 10.99 10.82 10.06 9.07 6.96 11.01 0.0 D11S1318 10.30 10.29 10.13 9.40 8.47 6.50 10.30 0.0 KVLQT1 14.19 14.17 13.94 12.89 11.54 8.68 14.19 0.0 D11S454 11.06 11.05 10.89 10.16 9.17 7.01 11.06 0.0 D11S860 5.77 6.92 8.32 9.14 8.92 7.46 9.15 0.058 D11S12 1.50 2.26 3.12 3.46 3.27 2.49 3.46 0.047 LOD scores were calculated with assumptions of 90% penetration and 0.001 gene frequency (ref 31). ^* Z _max represents the maximum LOD score and + θ _max represents the recombination rate evaluated at Z _max .

Example 8

Identification and Characterization of KVLQT1

Exon amplification by clones was performed from the physical map to identify candidate genes for LQT1. Exon trapping was performed using pSPL3B (Burn et al., 1995) on genomic P1 clones as described in Buckler et al., 1991; Church et al., 1994. 128 trapped exon minimums from each P1 clone were initially characterized by size of PCR product. From these, 400 clones were identified as A.L.F. Further analysis was made by dideoxy sequencing using an automated sequencer (Pharmacia). DNA sequence and database analysis showed eight possible exons with expected amino acid sequences that are similar to ion channels. The most similarity was obtained for 238 base pair trapped exons (4181A) with 53% similarity to potassium channel proteins from multiple species, including similarities to some of the putative pore regions. PCR analysis was used to map 4181A to the two P1s (118A10, 18B12) on the short arm of chromosome 11 and also from the physical map. This data indicates that 4181A is part of the potassium channel gene on chromosome 11p15.5.

Two different cDNA library screening methods were used to determine that trapped exon 4181A was part of the gene. Typical plaque filter hybridization with adult human cardiac cDNA libraries led to the identification of single positive clones. CDNA selection was changed to screen a second cardiac cDNA library (GENETRAPPER® cDNA Positive Selection System, Life Technologies, Inc.) and 12 independent clones were recovered. DNA sequence analysis showed complete alignment with the sequences derived from 4181A and the other trapped exons described above. The complex sequence of the cDNA clone is shown in Figure 3A. The longest open reading frame spans 1654 base pairs. Two coincident polyadenylation signals confirmed upstream of the poly (A) tail in the 3 ′ untranslated region. The identity of this initiation codon is not yet clear.

cDNA predicted proteins with structural features of potassium channels. Hydrotherapy analysis presents the phases of six major hydrophobic regions that can exhibit membrane spanning α-helices. This region is sequence similar to the potassium channel transmembrane domains S1-S6. A comparison of the identified genes and predicted amino acid sequences derived from Shaker (SHA) potassium channels (Pongs et al., 1988) is shown in FIG. 3B. In the region containing S1-S6, amino acid sequence identity was 30% and similarity was 59%. The sequence located 3 'of S1-S6 did not have significant similarity with any known protein. Since this gene is very similar to the voltage gate controlled potassium channel gene and is a strong candidate for LQT1, it was named KVLQT1.

Northern blot analysis was used to confirm the tissue distribution of KVLQT1 mRNA. KVLQT1 cDNA probe detected 3.2 kb transcripts in human heart, kidney, lung and placenta, but not skeletal muscle, liver or brain (FIG. 4). The heart showed the highest KVLQT1 mRNA.

Example 9

Characterization of the Complete KVLQT1 cDNA

The above studies resulted in the cloning and characterization of incomplete cDNA of KVLQT1. The sequence of this incomplete cDNA predicted a protein with six hydrophobic membrane spanning α-helix (S1-S6) and a typical K + channel pore display sequence (Heginbotham et al., 1994). However, this cDNA seems to have lost the amino terminal domain and has not functionally expressed. Several cDNA libraries were screened and new clones isolated to define the complete sequence of KVLQT1. Screening was performed by radiolabeling the partial KVLQT1 cDNA at ³² P and screening several cDNA libraries purchased from Clontech. 1.2 kb clones were isolated from the pancreatic library, subcolonized with pBluescript II and sequenced. This clone contained an in-frame ATG sequence with putative translational start site and original KVLQT1 clone. This new sequence data was combined with the initial sequence data to obtain cDNA sequences encoding the complete protein. cDNA sequences and 5 ′ and 3 ′ untranslated regions are shown in FIGS. 12A-12D. The new cDNA sequence predicted a 581 amino acid protein with a complete S1 domain and 27 amino acid N-terminal region. This is shown in Figure 8A. To demonstrate that this new sequence was part of the chromosome 11p15.5 bound KVLQT1 gene, hybridization experiments with DNA from a panel of somatic hybrids using 135 base pair XhoI restriction fragments from this region were performed. The new 5 'end was mapped to the short arm of chromosome 11. Northern analysis using the new KVLQT1 sequence showed hybridization with 3.2 kb of single messenger RNA in human pancreas, heart, kidney, lung and placenta, but not in brain, liver or skeletal muscle (FIG. 8B). Northern analysis was performed using a multi-tissue northern filter (Human MTN blot 1, Clontech) as shown in Curran et al., 1995.

Example 10

Characterization of the KVLQT1 Function

To limit the function of KVLQT1, Chinese hamster ovary (CHO) cells were transfected with the complete cDNA described in Example 9. KVLQT1 cDNA was subcloned into pCEP4 (InVitrogen). CHO cells were cultured in Ham's F-12 medium and transiently transfected with lipofectamine (Gibco BRL). Cells were transfected for 18 h in a 35 mm dish containing 6 μL lipofectamine, 0.5 μg green fluorescent protein (pGreen Lantern-1, Gibco BRL) and 1.5 μg KVLQT1 in pCEP4. 48-78 hours after transfection of fluorescent cells, voltage clamping was performed using an Axopatch 200 patch clamp amplifier (Axon Instruments). The bath solution contained 142 NaCl, 2 KCl, 1.2 MgCl ₂ , 1.8 CaCl ₂ , 11.1 glucose, 5.5 HEPES buffer (pH 7.4, 22-25 ° C.) in mM. The pipette solution contained 110 potassium glutamate, 20 KCl, 1.0 MgCl ₂ , 5 EGTA, 5 K ₂ ATP, 10 HEPES (pH 7.3) in mM. Data acquisition and analysis was performed using pCLAMP6 (Axon Instruments). The voltage dependence of current activation was determined by establishing a relationship between the tail current (determined by extrapolation to the end of the test pulse on the inactivation phase of the current) and the test potential with the Boltzmann function. Tail currents were normalized to the largest value for each oocyte.

The voltage dependent external K ⁺ currents were observed after membrane depolarization at potentials above −60 mV (FIG. 9A). This current reached steady state within 1 second at +40 mV. A simple delay was made prior to activation of the current, and repolarization to -70 mV induced tail currents with an initial increase in amplitude (hook) before deactivation. Similar tail current hooks were observed for HERG K ⁺ channels in advance and were due to channel recovery from inactivation at a faster rate than inactivation (Sanguinetti et al., 1995; Smith et al., 1996; Spector et al. ., 1996). Activation curve for KVLQT1 current was half maximum -11.6 (V _½) from ± 0.6 mV, 12.6 ± 0.5 mV in the slope factor; had a (n = 6 Fig. 9B).

The biophysical properties of KVLQT1 differed from other known cardiac K ⁺ currents. It was assumed that KVLQT1 associates with other subunits to form a known cardiac channel. The slow activation delayed rectifier K ⁺ current, I _Ks , regulates the repolarization of the cardiac action potential. Despite intensive research, the molecular structure of the I _Ks channel is not understood. Physiological data is 130 amino acid protein (Takumi et al, 1988.) Is minK (Goldstein and Miller, 1991 is a component of the I _Ks channel with a single estimate transmembrane domain; Hausdorff et al, 1991;. Takumi et al,. 1991; Busch et al., 1992; Wang and Goldstein, 1995; Wang et al., 1996). However, the size and structure of this protein raise questions about whether minK alone forms a functional channel (Attali et al., 1993; Lesage et al., 1993).

To test this hypothesis, CHO cells were cotransfected with KVLQT1 and human minK (hminK) cDNAs. hminK cDNA was subcloned into pCEP4 (InVitrogen) and transfection was performed as described above for KVLQT1. 0.75 μg each cDNA was used for simultaneous transfection of KVLQT1 and hminK. As previously reported (Lesage et al., 1993), transfection of CHO cells with hminK alone did not induce a detectable current (n = 10, FIG. 9C). Simultaneous transfection of KVLQT1 and hminK resulted in much slower activation delayed rectifier current than currents in cells transfected with KVLQT1 alone (FIGS. 9D and 9E). A delay of several hundred msec was sustained prior to the slow activation of the current of co-transfected CHO cells, indicating that there was no significant homomer KVLQT1 channel current. The current did not saturate during long-term depolarization pulses and requires a third order function that best describes the initial delay and the ideal of current activation. During a 30 second depolarization pulse to +40 mV, the current was activated with time constants of 0.68 ± 0.18, 1.48 ± 0.16 and 8.0 ± 0.6 seconds (n = 4). The isochronous (7.5 sec) activation curve for the current had a V _½ of 7.5 ± 0.9 mV and a slope factor of 16.5 ± 0.8 mV (n = 7; FIG. 9B). For comparison, the V _½ and slope of the activation curve for human heart I _Ks were 9.4 mV and 11.8 mV (Li et al., 1996). As KVLQT1 and hminK coexpress in CHO cells, activation of cardiac I _Ks is extremely slow and best explained by cubic function (Balser et al., 1990; Sanguinetti and Jurkiewicz, 1990). Quinidine (50 μM) blocked tail current by 30 ± 8% (n = 5) in cotransfected CHO cells, similar to its effect on I _Ks of isolated myocytes (40-50% blocking) ( Balser et al., 1991). Thus, simultaneous expression of KVLQT1 and hminK in CHO cells induced K ⁺ currents with biophysical properties nearly identical to those of cardiac I _Ks .

To further characterize the properties of hminK and KVLQT1, these channels were isolated and also expressed together in Xenopus oocytes. Xenopus laevis oocytes were isolated and injected with cRNA as described in Sanguinetti et al., 1995. KVLQT1 cDNA was subcloned with pSP64 (Promega). HminK cDNA was donated from S. Swanson. Simultaneous infusion experiments were performed using approximately equimolar concentrations of KVLQT1 cDNA (5.8 ng per oocyte) and hminK (1 ng per oocyte) cRNA. The bath solution contained 98 NaCl, 2 KCl, 2 MgCl ₂ , 0.1 CaCl ₂ , and 5 HEPES (pH 7.6, 22-25 ° C.) in mM. For reverse potential experiments, osmotic pressure was maintained by equimolar substitution of external NaCl to KCl. Three days after injection of oocytes with cRNA, currents were recorded using standard two microelectrode voltage clamp techniques (Sanguinetti et al., 1995). The current was filtered at 0.5 kHz and digitized at 2 kHz. Data is shown as mean ± sem.

Oocytes injected with KVLQT1 complementary to RNA expressed rapid activation external K ⁺ currents with voltage dependence of activation almost identical to CHO cells transfected with KVLQT1 cDNA (FIGS. 10A and 10B). K ⁺ selectivity of the KVLQT1 channel was determined by measuring the reverse potential (E _rev ) of the tail current at different concentrations of extracellular K ([K ⁺ ] _e ). The slope of the relationship between E _rev and log [K ⁺ ] _e was 49.9 ± 0.4 mV (n = 7; FIG. 10C), predicted by the Nernst equation (58 mV) for the fully selective K ⁺ channel. Considerably less than that. Simultaneous injection of oocytes by KVLQT1 and hminK cRNA induced a current similar to I _Ks (FIG. 11C). The slope of the relationship between E _rev and log [K ⁺ ] _e for co-implanted oocytes was 49.9 ± 4 mV (n = 6), similar to that of KVLQT1 alone and guinea pig heart I _Ks (49 mV). (Matsuura et al., 1987). The simultaneous injection isochronous I (7.5 sec) activation curve for the cells had a V _½ of 6.2 mV similarly, and the slope (Fig. 11 E) of 12.3 mV and cardiac I _Ks.

Example 11

Identification of KVLQT1 Gene in Xenopus

In contrast to CHO cells, hminK was able to express functional expression in Xenus oocytes (FIG. 11B). The induced current (I _sK ) was less than that induced in co-implanted oocytes, but the kinetics and voltage dependence of activation were similar (FIG. 11 AE). Two observations led to the hypothesis that I _sK of Xenus oocytes was formed from channels formed by association of minK with subunits expressed as unidentified components. Initially, the amount of I _Ks saturated after injection of a very small amount of minK cRNA (FIG. 11D), suggesting that limited amounts of endogenous components are required for functional expression (Wang and Goldstein, 1995; Cui et al., 1994). Second, heterologous expression of minK in mammalian cells does not induce a detectable current (Lesage et al., 1993) (FIG. 9C), suggesting that minK is not sufficient to form a functional channel. It has been assumed that this unidentified subunit may be a homologue of KVLQT1. To test this hypothesis, Xenofus oocyte cDNA library (Clontech) was screened with KVLQT1 cDNA clones expanded to the S3-S5 domain. 1.6 kb partial clone (XKVLQT1, FIG. 8A) was isolated. XKVLQT1 is 88% identical in amino acid concentration with the corresponding region of KVLQT1 (FIG. 8A). This data suggests that I _sK is formed from the co-aggregation of XKVLQT1 and minK proteins.

It was concluded that KVLQT1 and hminK associate together to form a cardiac I _Ks channel. Two delayed rectifiers K ⁺ currents, I _Kr and I _Ks , regulate the duration of action potentials in cardiomyocytes (Li et al., 1996; Sanguinetti and Jurkiewicz, 1990). Previous reports have associated abnormalities in I _Kr channels with long-term QT syndrome (Sanguinetti et al., 1995; Curran et al., 1995; Sanguinetti et al., 1996). The observation that KVLQT1 mutations also cause this disease (Wang et al., 1996) and the finding that KVLQT1 forms part of I _Ks and channels suggests that abnormalities in both cardiac delayed rectifier K ⁺ channels contribute to the risk of sudden cardiac arrhythmias Indicates that

Example 12

Simultaneous Isolation of LQT and KVLQT1 Missense Mutations from Six Large Families

To test the hypothesis that KVLQT1 is LQT1, affected members of K1532, the largest LQT family showing linkage to chromosome 11, were screened for functional mutations using single strand morphology polymorphism (SSCP) analysis. SSCP was performed as previously described (Wang et al., 1995a; Wang et al., 1995b). Normal and abnormal SSCP products were isolated and sequenced directly as described in Wang and Keating, 1994 or subcloned with pBluescript (SK +; Stratagene) using the T-vector method (Marchuk et al., 1990). When using the latter method, several clones were sequenced by dideoxy chain termination method using Sequenase® version 2.0 (United States Biochemicals, Inc.). The analysis was concentrated in the area between S2 and S6 because this area is important for KVLQT1 function. We designed oligonucleotide primers based on cDNA sequences and used these primers for cycle sequencing reactions with KVLQT1-containing P1, 18B12 (Wang and Keating, 1994). This experiment defined intron sequence flanking exons encoding S2-S6. Subsequently, additional primers were generated from this intron sequence and used for SSCP analysis (Table 2).

SSCP analysis identified anomalous isoforms in 70 affected members of K1532 (FIG. 5). Such aberrant isoforms were not observed in 147 unaffected members of this family or in genomic DNA obtained from 200 or more unrelated control individuals (Q. Wang, private data). The two point LOD score for the link between this variant and LQT was 14.19 at recombination rate (Table 1). No recombination was observed between KVLQT1 and LQT1, indicating that this site is fully bound. DNA sequence analysis of normal and abnormal SSCP isoforms showed a single base substitution, G to A transition from the first nucleotide of codon Val-125 (FIG. 5 and Table 3). This mutation results in the substitution of valine for methionine in the predicted intracellular domain between S4 and S5.

To further test the hypothesis that mutations in KVLQT1 cause LQT, DNA samples from affected members of an additional large five LQT family were studied. Linkage analysis by polymorphic markers from this region indicated that the disease phenotype is linked to chromosome 11 in these families (Q. Wang, undisclosed results). The above SSCP isoforms were identified in affected members of K1723, K2605, K1807 (FIG. 5), K161 and K162 (Q. Wang, closed results). SSCP variants identified in K161 and K162 were the same as those observed in K1807 (Q. Wang, closed results). No abnormal SSCP isoforms were observed in DNA samples obtained from unaffected members of these families or more than 200 unrelated control individuals (FIG. 5; Q. Wang, Private Data). Normal and abnormal isoforms identified in each family were sequenced. Nucleotide changes, coding effects and the location of each mutation are summarized in Table 3.

PCR primers used to define KVLQT1 mutations primer order Amplified area Sequence number One S2-S3 3 2 4 3 S3-S4 5 4 6 5 S4 7 6 8 7 S5-void 9 8 10 9 Air gap-S6 11 10 12 11 S6 13 12 14

Summary of KVLQT1 Mutants Codon Nucleotide changes Coding effect Mutation domain ancestry Affected numbers 38-39 ΔTCG fruition F38W / G39Δ S2 K13216 One 49 GCC to CCC Miss Sense A49P S2-S3 K13119 One 60 GGG to AGG Miss Sense G60R S2-S3 K2557 3 61 CGG to CAG Miss Sense R61Q S2-S3 K15019 2 125 ATG to GTG Miss Sense V125M S4-S5 K1532 70 144 CTC to TTC Miss Sense L144F S5 K1777 2 177 GGG to AGG Miss Sense G177R air gap K20926 One 183 ACC to ATC Miss Sense T1831 air gap K20925 One 212 GAG to GCG Miss Sense A212E S6 K1723 6 212 GAG to GCG Miss Sense A212E S6 K2050 2 212 GTG to GCG Miss Sense A212V S6 K1807 6 212 GTG to GCG Miss Sense A212V S6 K161 18 212 GTG to GCG Miss Sense A212V S6 K162 18 212 GTG to GCG Miss Sense A212V S6 K163 3 212 GTG to GCG Miss Sense A212V S6 K164 2 216 GGG to GAG Miss Sense G216E S6 K2605 11

Example 13

KVLQT1 Gene Deletion and Nine Missense Mutations Associated with LQT in Small Family and Sporadic Cases

To further identify LQT related mutations in KVLQT1, SSCP analysis was performed on small household and sporadic cases. SSCP found abnormal heteromorphs in the household 13216 (FIG. 6). Analysis of 200 or more unrelated control subjects did not show this heterogeneity (Q. Wang, Private Data). Cloning and sequencing the above heterologs revealed three base pair deletions, including codons 38 and 39. This mutation resulted in the substitution of phenylalanine for tryptophan and deletion of glycine in the putative S2 domain (Table 3).

The above SSCP isoforms have been identified in affected members of an additional nine families: K1777, K20925, K13119, K20926, K15019 (Figure 6), K2050, K163 and K164 (Q. Wang, Private Data). The aberrant SSCP isoforms identified in K2050 were the same as those observed in K1723, and the aberrant isomers identified in K163 and K164 were the same as those observed in K1807. Abnormal isoforms were not observed in DNA samples from more than 200 control subjects (Q. Wang, Private Data). In each case, normal and abnormal isoforms were sequenced. This data is shown in FIG. 6 and summarized in Table 3. Overall, KVLQT1 mutations associated with LQT in 16 family or sporadic cases were identified (FIG. 7), providing strong molecular genetic evidence that mutations in KVLQT1 result in chromosome 11-linked forms of LQT.

Example 14

Formation of Polyclonal Antibodies to KVLQT1

KVLQT1 coding sequence fragment as E. coli. It was expressed as a fusion protein in Collie. Overexpressed proteins were purified by gel elution and using them to immunize rabbits and mice following procedures similar to those described in Harlow and Lane, 1988. This procedure has been shown to generate Abs for a variety of different proteins (eg, Kraemer et al., 1993).

Briefly, KVLQT1 coding sequence stretch was cloned as fusion protein in plasmid PET5A (Novagen, Inc., Madison, Wis.). After induction with IPTG, overexpression of the fusion protein with the expected molecular weight was verified by SDS / PAGE. Fusion proteins were purified from gels by electroelutation. Identification of the protein as a KVLQT1 fusion product was demonstrated by protein sequencing at the N-terminus. Next, purified protein was used as immunogen in rabbits. Rabbits were immunized with 100 μg of protein in complete Freund's supplement and twice at three week intervals, initially with 100 μg of immunogen in incomplete Freund's supplement, followed by 100 μg of immunogen in PBS. Antibody containing serum was collected two weeks thereafter.

This procedure was repeated to form antibodies against the mutant form of the KVLQT1 gene. This antibody was used in conjunction with an antibody against wild type KVLQT1 to detect the presence and relative concentration of mutant forms in various tissues and biological fluids.

Example 15

Formation of Monoclonal Antibodies Specific for KVLQT1

Monoclonal antibodies were formed according to the following procedure. Mice were immunized with an immunogen consisting of complete KVLQT1 or KVLQT1 peptide (wild type or mutant) conjugated to keyhole impetus hemocyanin using glutaraldehyde or EDC as is well known in the art.

Immunogen was mixed with adjuvant. Each mouse was injected four times with 10-100 μg of immunogen, and after the fourth injection, blood samples were taken from the mice to confirm that the serum contained antibodies to the immunogen. Serum titers were determined by ELISA or RIA. Mice with sera indicating the presence of antibodies to the immunogen were selected for hybridoma production.

Spleens were removed from immune mice and single cell suspensions were prepared (Harlow and Lane, 1988). Cell fusion was performed as almost described (Kohler and Milstein, 1975). Briefly, P3.65.3 myeloma cells (American Type Culture Collection, Rockville, MD) were fused with immune spleen cells using polyethylene glycol as described in Harlow and Lane, 1988. Cells were plated at a density of 2 × 10 ⁵ cells / well in 96 well tissue culture plates. Individual wells were examined for growth and the presence of KVLQT1 specific antibodies was tested by ELISA or RIA using wild type or mutant KVLQT1 target proteins against the supernatant of grown wells. Cells in positive wells were expanded and subcloned to confirm monoclonal formation.

Clones with the desired specificity were expanded and grown as ascites in mice or hollow fiber systems to form sufficient antibodies for characterization and assay development.

Example 16

Sandwich Analysis for KVLQT1

Monoclonal antibodies were attached to plates, tubes, beads or granules and solid surfaces. Preferably, the antibody is attached to the well surface of a 96-well ELISA plate. 100 μl samples (eg serum, urine, tissue cytosol) containing KVLQT1 peptide / protein (wild type or mutant) were added to the solid antibody. This sample was incubated for 2 hours at room temperature. The sample fluid was then decanted and the solid phase was washed with buffer to remove unbound material. 100 μl of the second monoclonal antibody (for other genes on the KVLQT1 peptide / protein) was added to the solid phase. This antibody was labeled with a detection molecule (eg 125-I, enzyme, fluorophore or chromophore) and the solid phase with the second antibody was incubated for 2 hours at room temperature. The second antibody was decanted and the solid phase was washed with buffer to remove unbound material.

The amount of binding label was quantified in proportion to the amount of KVLQT1 peptide / protein present in the sample. Individual analyzes were performed using monoclonal antibodies specific for wild type KVLQT1 and monoclonal antibodies specific for each mutation identified in KVLQT1.

Example 17

Screening Drug Analysis Influencing KVLQT1 and minK K ⁺ Channels

Now, with the knowledge that KVLQT1 and minK associate together to form a cardiac I _Ks potassium channel, one can invent an assay that screens for drugs that will have an effect on these channels. Two genes, KVLQT1 and minK, were cotransfected into oocytes or mammalian cells and co-expressed as described above. Cotransfection was carried out using any combination of wild type or specifically mutated KVLQT1 and minK. When one of the genes used for co-transfection contains a mutation that causes LQT, the induced current change can be seen compared to co-transfection with only the wild-type gene. Drug candidates were added to bath solutions of transfected cells to test the effect of drug candidates on induced currents. Drug candidates that change the induced current to be closer to the current known by cells co-transfected with wild type KVLQT1 and minK are useful for treating LQT.

Although the present invention has been disclosed in this patent application with reference to preferred embodiments, the description is not to be taken in a limiting sense, but as an example, and is easily modified by those skilled in the art within the spirit of the invention and the scope of the appended claims. It is expected to be.

references

European Patent Application Publication No. 0332435

European Patent Application Publication No. 225,807

EP73,675A to Hitzeman et al.

Patents and patent applications

PCT Application Publication WO 93/07282

U.S. Patent 4,376,110

U.S. Patent 4,486,530

U.S. Patent 4,868,105

U.S. Patent 5,252,479

<Sequence table>

(1) General Information:

(i) Applicant: University of Utah Research Foundation

(ii) Name of the Invention: Long-term QT syndrome gene encoding KVLQT1, which associates with ＫＶＬＱＴ1-minK to form a heart I _KS potassium channel

(iii) SEQ ID NO: 26

(iv) communication address:

(A) Recipients: Venable, Battier, Howard & Savileti, Ellp

(B) Street: Suite 1000 yen. New York Avenue 1201

(C) City: Washington

(D) Note: DC

(E) Country: United States

(F) ZIP Code: 20005

(v) computer-readable form

(A) Media Type: Floppy Diskette

(B) Computer: IBM PC compatible

(C) working system: PC-DOS / MS-DOS

(D) Software: Word 6.0 for Windows

(vi) Current application data:

(A) Application number: -Undecided-

(B) Application date: December 20, 1996

(C) Classification:

(vii) Attorney / Agent Information:

(A) Statement: Inen, Jeffrey L.

(B) registration number: 28,957

(C) Reference / Event No .: 19780-116871-WO

(ix) Communications Information:

(A) Phone: 202-962-4800

(B) Fax: 206-962-8300

(2) Information about SEQ ID NO: 1

(i) Sequence Features:

(A) Length: 17 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "Synthetic Oligomer"

(xi) sequence description: SEQ ID NO: 1

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 17 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "Synthetic Oligomer"

(xi) sequence description: SEQ ID NO: 2

(2) information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 22 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) sequence description: SEQ ID NO: 3:

(2) information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 21 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) sequence description: SEQ ID NO: 4

(2) information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 20 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) sequence description: SEQ ID NO: 5:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 22 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) Sequence description: SEQ ID NO: 6:

(2) information for SEQ ID NO: 7

(i) Sequence Features:

(A) Length: 20 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) sequence description: SEQ ID NO: 7:

(2) information for SEQ ID NO: 8:

(i) Sequence Features:

(A) Length: 20 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) sequence description: SEQ ID NO: 8

(2) information for SEQ ID NO: 9:

(i) Sequence Features:

(A) Length: 22 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) Sequence description: SEQ ID NO: 9:

(2) Information about SEQ ID NO: 10:

(i) Sequence Features:

(A) Length: 22 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) sequence description: SEQ ID NO: 10:

(2) information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 22 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) sequence description: SEQ ID NO: 11:

(2) information for SEQ ID NO: 12:

(i) Sequence Features:

(A) Length: 20 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) SEQ ID NO: 12:

(2) information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 21 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) sequence description: SEQ ID NO: 13:

(2) information about SEQ ID NO: 14:

(i) Sequence Features:

(A) Length: 24 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: other nucleic acids

(A) Description: / desc = "PCR primer"

(xi) SEQ ID NO: SEQ ID NO: 14

(2) Information about SEQ ID NO: 15:

(i) Sequence Features:

(A) Length: 2633 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) Molecular Form: cDNA

(iii) hypothetical: no

(iv) antisense: no

(vi) source:

(A) Creature: Homo sapiens

(ix) Features:

(A) Name / symbol: CDS

(B) Location: 2..1642

(xi) SEQ ID NO: 15:

(2) information for SEQ ID NO: 16:

(i) Sequence Features:

(A) Length: 547 amino acids

(B) Type: Amino Acid

(D) topology: linear

(ii) Molecular Form: Protein

(iii) hypothetical: no

(xi) sequence description: SEQ ID NO: 16:

(2) information for SEQ ID NO: 17:

(i) Sequence Features:

(A) Length: 26 amino acids

(B) Type: Amino Acid

(C) strands: single

(D) topology: linear

(ii) molecular form: peptide

(iii) hypothetical: no

(xi) SEQ ID NO: 17:

(2) information for SEQ ID NO: 18:

(i) Sequence Features:

(A) Length: 61 amino acids

(B) Type: Amino Acid

(C) strands: single

(D) topology: linear

(ii) molecular form: peptide

(iii) hypothetical: no

(xi) sequence description: SEQ ID NO: 18

(2) information for SEQ ID NO: 19:

(i) Sequence Features:

(A) Length: 137 amino acids

(B) Type: Amino Acid

(C) strands: single

(D) topology: linear

(ii) molecular form: peptide

(iii) hypothetical: no

(xi) sequence description: SEQ ID NO: 19:

(2) information about SEQ ID NO: 20:

(i) Sequence Features:

(A) Length: 66 amino acids

(B) Type: Amino Acid

(C) strands: single

(D) topology: linear

(ii) molecular form: peptide

(iii) hypothetical: no

(xi) sequence description: SEQ ID NO: 20:

(2) information about SEQ ID NO: 21:

(i) Sequence Features:

(A) Length: 123 amino acids

(B) Type: Amino Acid

(C) strands: single

(D) topology: linear

(ii) molecular form: peptide

(iii) hypothetical: no

(xi) sequence description: SEQ ID NO: 21:

(2) information for SEQ ID NO: 22:

(i) Sequence Features:

(A) Length: 58 amino acids

(B) Type: Amino Acid

(C) strands: single

(D) topology: linear

(ii) molecular form: peptide

(iii) hypothetical: no

(xi) sequence description: SEQ ID NO: 22:

(2) information for SEQ ID NO: 23:

(i) Sequence Features:

(A) Length: 376 amino acids

(B) Type: Amino Acid

(C) strands: single

(D) topology: linear

(ii) Molecular Form: Protein

(iii) hypothetical: no

(iv) Source:

(A) Creature: Xeropherus

(xi) sequence description: SEQ ID NO: 23:

(2) information for SEQ ID NO: 24:

(i) Sequence Features:

(A) Length: 581 amino acids

(B) Type: Amino Acid

(C) strands: single

(D) topology: linear

(ii) Molecular Form: Protein

(iii) hypothetical: no

(iv) Source:

(A) Creature: Homo sapiens

(xi) sequence description: SEQ ID NO: 24:

(2) information for SEQ ID NO: 25:

(i) Sequence Features:

(A) Length: 2821 base pairs

(B) Type: nucleic acid

(C) strands: double

(D) topology: linear

(ii) Molecular Form: DNA (Genome)

(iii) hypothetical: no

(iv) antisense: no

(vi) source:

(A) Creature: Homo sapiens

(ix) Features:

(A) Name / symbol: CDS

(B) Location: 88..1830

(xi) SEQ ID NO: 25:

(2) information for SEQ ID NO: 26:

(i) Sequence Features:

(A) Length: 581 amino acids

(B) Type: Amino Acid

(D) topology: linear

(ii) Molecular Form: Protein

(xi) sequence description: SEQ ID NO: 26:

Claims (30)

Cells transfected with DNA encoding human minK. Cells transfected with RNA complementary to DNA encoding human minK. The cell of claim 1 further transfected with DNA encoding human KVLQT1. The cell of claim 1 further transfected with DNA encoding a mutant human KVLQT1. The cell of claim 2, further transfected with RNA complementary to DNA encoding human KVLQT1. The cell of claim 2, further transfected with RNA complementary to DNA encoding a mutant human KVLQT1. Cells transfected with DNA encoding the mutant human minK. Cells transfected with RNA complementary to DNA encoding mutant human minK. The cell of claim 7 further transfected with DNA encoding human KVLQT1. The cell of claim 8 further transfected with RNA complementary to DNA encoding human KVLQT1. a) introducing the cells of claim 3 or 5 into a bathing solution to measure current; b) measuring the K ⁺ current induced in the cells of step (a); c) introducing the cells of claim 4 or 6 into a bath solution to measure current; d) measuring the K ⁺ currents induced in the cells of step (c); e) adding the drug to the bath solution of step (d); f) measuring the K ⁺ current induced in the cells of step (e); g) the drug is more or less similar to the induced K ⁺ current measured in cells co-transfected with wild-type minK and KVLQT1 compared to the current measured in cells co-transfected with wild-type minK and mutant KVLQT1 in the absence of drug. Measuring whether or not to induce current A method for screening a drug useful for treating long-term QT syndrome, comprising: generating a current more similar to the current measured in cells co-transfected with wild-type minK and KVLQT1. a) introducing the cells of claims 3 or 5 into the bath solution to measure current; b) measuring the K ⁺ current induced in the cells of step (a); c) introducing the cells of claim 9 or 10 into the bath solution to measure current; d) measuring the K ⁺ currents induced in the cells of step (c); e) adding the drug to the bath solution of step (d); f) measuring the K ⁺ current induced in the cells of step (e); g) Currents that are more or less similar to the induced K ⁺ currents measured in cells co-transfected with wild-type minK and KVLQT1 when compared to currents measured in cells cotransfected with mutations minK and KVLQT1 in the absence of drug. Measuring whether or not A method for screening a drug useful for treating long-term QT syndrome, comprising: generating a current more similar to the current measured in cells co-transfected with wild-type minK and KVLQT1. a) introducing the cells of claims 3 or 5 into the bath solution to measure current; b) measuring the K ⁺ current induced in the cells of step (a); c) introducing the cells of claim 4 or 6 into a bath solution to measure current; d) measuring the K ⁺ currents induced in the cells of step (c); e) adding the drug to the bath solution of step (d); f) measuring the K ⁺ current induced in the cells of step (e); g) the drug is more or less similar to the induced K ⁺ current measured in cells co-transfected with wild-type minK and KVLQT1 compared to the current measured in cells co-transfected with wild-type minK and mutant KVLQT1 in the absence of drug. Measuring whether or not to induce current A method for screening a drug useful for long-term QT syndrome prevention, comprising: generating a current more similar to the current measured in cells co-transfected with wild-type minK and KVLQT1. a) introducing the cells of claims 3 or 5 into the bath solution to measure current; b) measuring the K ⁺ current induced in the cells of step (a); c) introducing the cells of claim 9 or 10 into the bath solution to measure current; d) measuring the K ⁺ currents induced in the cells of step (c); e) adding the drug to the bath solution of step (d); f) measuring the K ⁺ current induced in the cells of step (e); g) Currents that are more or less similar to the induced K ⁺ currents measured in cells co-transfected with wild-type minK and KVLQT1 when compared to currents measured in cells cotransfected with mutations minK and KVLQT1 in the absence of drug. Measuring whether or not A method for screening a drug useful for long-term QT syndrome prevention, comprising: generating a current more similar to the current measured in cells co-transfected with wild-type minK and KVLQT1. The method of claim 11, wherein the cell is a mammalian cell. The method of claim 11, wherein the cell is a CHO cell. Transgenic animals other than humans, including wild type human minK. The animal of claim 17, further comprising human KVLQT1. The animal of claim 17, further comprising a mutant human KVLQT1. A transgenic animal other than human, comprising a mutant human minK. The animal of claim 20, further comprising human KVLQT1. a) measuring the K ⁺ current induced in the transgenic animal of claim 18; b) measuring the K ⁺ current induced in the transgenic animal of claim 21; c) administering the drug to the transgenic animal of step (b); d) measuring the K ⁺ currents induced in the drug treated animal of step (c); e) the drug is more or less similar to the induced K ⁺ current measured in cells co-transfected with wild-type minK and KVLQT1 compared to the current measured in cells co-transfected with mutant minK and mutant KVLQT1 in the absence of drug. Measuring whether or not to induce current A method for screening a drug useful for treating long-term QT syndrome, comprising: generating a current more similar to the current measured in cells co-transfected with wild-type minK and KVLQT1. a) measuring the K ⁺ current induced in the transgenic animal of claim 18; b) measuring the K ⁺ currents induced in the transgenic animal of claim 19; c) administering the drug to the transgenic animal of step (b); d) measuring the K ⁺ currents induced in the drug treated animal of step (c); e) the drug is more or less similar to the induced K ⁺ current measured in cells co-transfected with wild-type minK and KVLQT1 compared to the current measured in cells co-transfected with wild-type minK and mutant KVLQT1 in the absence of the drug. Measuring whether or not to induce current A method for screening a drug useful for treating long-term QT syndrome, comprising: generating a current more similar to the current measured in cells co-transfected with wild-type minK and KVLQT1. a) measuring the K ⁺ current induced in the transgenic animal of claim 18; b) measuring the K ⁺ current induced in the transgenic animal of claim 21; c) administering the drug to the transgenic animal of step (b); d) measuring the K ⁺ currents induced in the drug treated animal of step (c); e) A current that is more or less similar to the induced K ⁺ current measured in cells co-transfected with wild-type minK and KVLQT1 compared to the current measured in cells co-transfected with minK and mutant KVLQT1 in the absence of drug. Measuring whether or not A method for screening a drug useful for long-term QT syndrome prevention, comprising: generating a current more similar to the current measured in cells co-transfected with wild-type minK and KVLQT1. a) measuring the K ⁺ current induced in the transgenic animal of claim 18; b) measuring the K ⁺ currents induced in the transgenic animal of claim 19; c) administering the drug to the transgenic animal of step (b); d) measuring the K ⁺ currents induced in the drug treated animal of step (c); e) the drug is more or less similar to the induced K ⁺ current measured in cells co-transfected with wild-type minK and KVLQT1 compared to the current measured in cells co-transfected with wild-type minK and mutant KVLQT1 in the absence of the drug. Measuring whether or not to induce current A method for screening a drug useful for long-term QT syndrome prevention, comprising: generating a current more similar to the current measured in cells co-transfected with wild-type minK and KVLQT1. An isolated nucleic acid or complement thereof encoding a Xenopus KVLQT1 polypeptide having the amino acid sequence set forth in SEQ ID NO: 23. An isolated wild type Xenopus KVLQT1 polypeptide having the amino acid sequence set forth in SEQ ID NO: 23. By comparing the sequence of the minK gene or its expression product isolated from a tissue sample of human assessment with the wild-type minK gene or its expression product, the minK gene in the sequence of evaluation to be indicative of the risk of long-term QT syndrome for the evaluation subject. A method of assessing the risk of a human assessment target for long-term QT syndrome, which comprises screening for mutations in the. The method of claim 28, wherein said expression product is selected from the group consisting of mRNA of minK gene and minK polypeptide encoded by minK gene. The method of claim 28 or 29, (a) observe the change in mobility of electrophoresis of single stranded DNA from the sample on an unmodified polyacrylamide gel, (b) hybridizing the probe to the DNA under conditions suitable to hybridize minK gene probe to genomic DNA isolated from the sample, (c) measuring hybridization of allele specific probes to genomic DNA isolated from the sample, (d) amplifying all or part of the minK gene isolated from the sample to form an amplified sequence and sequencing the amplified sequence, (e) the presence of specific minK mutant alleles in the sample is determined by nucleic acid amplification, (f) molecular cloning all or part of the minK gene isolated from the sample to form a cloned sequence and sequencing the cloned sequence, (g) the molecule (1) and the molecule (1) when the minK gene genomic DNA or minK mRNA isolated from the sample, and (2) nucleic acid probes complementary to the human wild type minK gene DNA hybridize with each other to form a duplex; 2) measure if there is a mismatch between them, (h) amplifying the minK gene sequence in the sample and hybridizing the amplified sequence to a nucleic acid probe consisting of wild type minK gene sequence, (i) amplifying the minK gene sequence in the tissue and hybridizing the amplified sequence to a nucleic acid probe consisting of a mutant minK gene sequence, (j) screen for deletion mutations, (k) screen for point mutations, (l) screening for insertion mutations, (m) measuring in situ hybridization of the minK gene sequence or the minK gene in the sample with one or more nucleic acid probes consisting of a mutant minK gene sequence, (n) immunoblotting, (o) immunocytochemical treatment, (p) assay for binding interactions between binding partners capable of specifically binding to polypeptide expression products of minK mutant alleles and / or binding partners for minK polypeptides and minK gene proteins isolated from the tissue, (q) assaying for inhibition of biochemical activity of said binding partner How to perform one or more procedures.