WO2011035239A1 - Kcnq1 et kcne2 dans la maladie thyrodienne - Google Patents

Kcnq1 et kcne2 dans la maladie thyrodienne Download PDF

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WO2011035239A1
WO2011035239A1 PCT/US2010/049488 US2010049488W WO2011035239A1 WO 2011035239 A1 WO2011035239 A1 WO 2011035239A1 US 2010049488 W US2010049488 W US 2010049488W WO 2011035239 A1 WO2011035239 A1 WO 2011035239A1
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kcne2
thyroid
pups
kcnql
dams
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Geoffrey Abbott
Torsten Roepke
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Cornell University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy

Definitions

  • KCNQl is a voltage-gated K + channel a subunit noted for its key role in human ventricular repolarization, because it generates the ventricular, slowly activating, delayed rectifier K + current (I Ks ) by assembling with the KCNEl (originally named MinK) single-transmembrane domain ⁇ subunit 1 4 .
  • KCNEl belongs to a family of five proteins including KCNE2 (originally named MiRPl), which, like KCNEl, can regulate KCNQl and other a subunits such as hERG, often endowing unique functional properties 5 7 .
  • Inherited mutations in the genes encoding KCNQl, hERG, KCNEl and KCNE2 are all associated with life-threatening cardiac arrhythmias, including long QT syndrome 3 ' 4 ' 6 ' 8 , and may have a role in atrial fibrillation 9 12 . These subunits are also expressed in a variety of other tissues, but the potential for cardiac effects secondary to their dysfunction in these other tissues has been little studied.
  • KCNQl is unique among the voltage-gated K + channel a subunits in that, by assembly with KCNE2 or KCNE3, it can form constitutively active, K + 'leak' channels. KCNQl can thereby facilitate background K + flux in some nonexcitable, polarized epithelial cell types. KCNQl and KCNE3 are thought to form a channel in the basolateral membrane of colonic crypt cells 13 , and KCNQl -KCNE2 channels support function of the H /K + ATPase in the apical membrane of parietal cells. Accordingly, disruption of Kcnql or Kcne2 in mice causes achlorhydria and gastric hyperplasia 14 ' 15 .
  • thyrocytes Analogous to parietal cells and colonic crypt cells in the gastrointestinal tract, thyrocytes are nonexcitable, polarized epithelial cells expressing ion transporters essential for the function of the thyroid gland.
  • the thyroid hormones T 3 and T 4 are crucial for normal growth and development of the fetus and newborn, as well as for regulation of metabolism in virtually all tissues at all ages. Because of the scarcity of iodine, an essential constituent of T 3 and T 4 , iodide ( ⁇ ) deficiency disorders are still prevalent in many areas of the world and are thus at the forefront of global health initiatives.
  • NIS-mediated ⁇ transport uses the downhill Na + gradient generated by the Na + /K + ATPase at the basolateral membrane of the thyrocyte.
  • the role of K + channels in the thyroid is unknown.
  • KCNE2 (originally recognized for their functional roles in repolarizing cardiac myocytes) form a constitutively active K + channel in thyrocytes and that Kcne2 is required for normal thyroid hormone biosynthesis.
  • Thyroid dysfunction is a global health concern, causing defects including neurodevelopmental disorders, dwarfism and cardiac arrhythmia.
  • potassium channel subunits KCNQ1 and KCNE2 form a thyroid-stimulating hormone- stimulated, constitutively active, thyrocyte K + channel required for normal thyroid hormone biosynthesis.
  • Targeted disruption of Kcne2 in mice impaired thyroid iodide accumulation up to eightfold, impaired maternal milk ejection, halved milk
  • mice had hypothyroidism, dwarfism, alopecia, goiter and cardiac abnormalities including hypertrophy, fibrosis, and reduced fractional shortening.
  • the alopecia, dwarfism and cardiac abnormalities were alleviated by triiodothyronine (T 3 ) and T 4 administration to pups, by supplementing dams with T 4 before and after they gave birth, or by feeding exclusively from Kcne2 +I+ dams; conversely, these symptoms were elicited in Kcne2 +I+ pups by feeding exclusively from Kcne2 ⁇ ' ⁇ dams.
  • FIG. 3 Kcne2 ⁇ ' ⁇ mice are hypothyroid and treatable with T 3 and T 4 or wild-type surrogacy, (a) Left, serum T 4 in Kcne2 +I+ and Kcne2 ⁇ ' ⁇ mice at 3 weeks of age. Right, serum TSH in Kcne2 +I+ and Kcne2 ⁇ ' ⁇ mice at 3 weeks of age. *P ⁇ 0.001 compared to Kcne2 +/+ by ANOVA. Numbers of mice are shown in parentheses. Error bars indicate s.e.m.
  • Error bars indicate s.e.m.
  • (e) Mean body mass at 3-6 weeks of age for pups from wild-type and Tcne2-disrupted crosses surrogated (Sgt), or treated (Tx) with T 3 and T 4 ] injection (P) or by T 4 supplementation of their mothers (D); n 9-23 pups per group. Error bars indicate s.e.m.
  • FIG. 4 KCNE2 and KCNQ 1 form a TSH-stimulated thyrocyte K + channel, (a) Immunofluorescence using antibodies raised against KCNE2, KCNQ1 and NIS in sections of human thyrocytes from individuals with thyroid hyperplasia. DAPI visualization of nuclei is shown in blue. Asterisks indicate colloid. Scale bars, 4 ⁇ .
  • n 2 sections
  • n 3 experiments
  • n 2 thyroids per genotype
  • (f) Electron micrographs of thyroid epithelium from adult K n 2 and Kcne2 ⁇ ' ⁇ mice (from Kcne2 +I ⁇ x Kcne2 +I ⁇ crosses). Scale bars, 2 ⁇ . Representative of n 2 thyroids per genotype, (g) Western
  • n 4 or 5 mice per group; error bars indicate s.e.m. *P ⁇ 0.05.
  • (e) Mean 124 accumulation (in ⁇ ) in thyroid and stomach from imaging as in d, measured as the maximum radioactivity in each tissue minus mean background count in each mouse, n 7-12 pups per time point per group; error bars indicate s.e.m. *P ⁇ 0.05.
  • Kcne2 ⁇ ' ⁇ pups from Kcne2 ⁇ ' ⁇ dams have cardiomegaly
  • anterior and posterior wall thickening are indicators of cardiac hypertrophy, which was probably the primary effect of Kcne2 deficiency; the left ventricular dilation and reduced fractional shortening probably arose from a compensatory response (that is, the Frank- Starling mechanism 20 ) to the impaired contractility resulting from sustained hypertrophy.
  • Ventricular myocytes of 3 -week-old Kcne2 ⁇ ' ⁇ pups from homozygous crosses had a twofold larger membrane capacitance than those from age-matched Kcne2 +I+ pups, also indicative of hypertrophy (defined as increased organ or tissue size due to an increase in the size of the constituent cells) (Fig. Id).
  • Kcne2 _/ ⁇ mice show dwarfism and alopecia
  • Kcne2 ⁇ ' ⁇ mice showed other gross abnormalities that were influenced by the maternal Kcne2 ⁇ ' ⁇ genotype.
  • Pups from Kcne2 ⁇ ' ⁇ dams showed 50% embryonic lethality, whether the sire was Kcne2 ⁇ ' ⁇ or Kcne2 +I ⁇ (Fig. 2a).
  • Maternal genotype was the determining factor in litter size: litters from Kcne2 +I ⁇ dams with Kcne2 ⁇ ' ⁇ sires were of normal size, and surviving pups in litters from both types of Kcne2 ⁇ ' ⁇ x Kcne2 +I ⁇ crosses showed an approximately mendelian distribution (Fig. 2a). Kcne2 ⁇ ' ⁇ pups from Kcne2 ⁇ ' ⁇ dams also showed severe dwarfism (Fig. 2b, c).
  • Kcne2 ⁇ ' ⁇ pups producing dwarfism due to slow growth of both long bones and vertebrae (Fig. 2c). This effect was also apparent from the presence of larger epiphyseal gaps and less ossification of the epiphyses in the large joints compared to those of Kcne2 +I+ pups.
  • the large joints of Kcne2 ⁇ ⁇ pups were irregularly shaped, fragmented and heterogeneously sclerotic, defects characteristic of slow multifocal ossification (Fig. 2c).
  • Kcne2 ⁇ ' ⁇ pups from homozygous crosses also showed marked alopecia of the trunk, which began at 1-2 weeks of age and peaked at 4-5 weeks (Fig. 2b,e— g).
  • Fig. 2b,e— g We also observed alopecia in aging Kcne2 ⁇ ' ⁇ mice from Kcne2 +I ⁇ x Kcne2 +I ⁇ crosses, initiating between the ears then spreading posterodorsally, with an abrupt loss of mature hair follicles at the transition zones (Fig. 2h-j).
  • Kcne2 ⁇ ⁇ phenotype is attenuated by TH or Kcne2+/+ milk
  • Serum T 4 and TSH concentrations were, however, normal in virgin 3- to 6-month-old Kcne2 ⁇ ' ⁇ mice from Kcne2 +I ⁇ x Kcne2 +I ⁇ crosses (Supplementary Fig. 2), consistent with growth, litter size, and cardiac morphology trends (see Fig. 2 and ref. 19).
  • T 4 and TSH concentrations were trending down and up, respectively, in Kcne2 ⁇ ' ⁇ mice bred from Kcne2 +I ⁇ x Kcne2 +I ⁇ crosses (Supplementary Fig. 2), consistent with a latent hypothyroidism and the late onset of alopecia (Fig. 2h), cardiac hypertrophy and fibrosis (Fig. lj).
  • Kcne2 +I ⁇ crosses had a 40% greater mean mass post-mortem than thyroid glands from age-matched Kcne2 +I+ mice bred from Kcne2 +I ⁇ x Kcne2 +I ⁇ crosses, while thyroid glands from age-matched Kcne2 +I ⁇ mice bred from Kcne2 +I ⁇ x Kcne2 +I ⁇ crosses had intermediate mean mass, indicative of goiter formation due to Kcne2 disruption (Fig. 3b). In contrast to virgin adult mice, pregnant Kcne2 ⁇ ' ⁇ dams showed a 62% reduced mean serum T 4 concentration than pregnant Kcne2 +I+ dams (Fig.
  • Kcne2 +I+ dams as soon as possible after birth and through to weaning, such that Kcne2 ⁇ ' ⁇ pups were fed exclusively by Kcne2 +/+ dams, would alleviate any of the observed abnormalities.
  • normal body weight was fully restored in Kcne2 ⁇ ' ⁇ pups from Kcne2 ⁇ ' ⁇ x Kcne2 ⁇ ' ⁇ crosses by surrogacy with K n 2 dams (Fig. 3d,e).
  • Kcne2 ⁇ ' ⁇ pups born and raised by Kcne2 ⁇ ' ⁇ dams and from Kcne2 ⁇ ' ⁇ sires showed markedly improved body weight by 3 weeks of age after T 3 and T 4
  • KCNE2 and KCNQl form a TSH-stimulated thyrocyte K + channel
  • KCNE2 forms heteromeric channels with the KCNQl K + channel a subunit in gastric epithelium 14 ' 15 ' 25 ' 26 .
  • KCNE2 and KCNQl are expressed in human (Fig. 4a) and mouse (Fig. 4b-d) thyroid glands (note that hyperplastic human thyroid tissue was used to permit better distinction between the apical and basolateral membranes).
  • KCNE2 and KCNQl partially co-localized with NIS, the basolateral membrane glycoprotein that mediates active ⁇ transport, the first step in thyroid hormone biosynthesis.
  • KCNQl -KCNE2 K + currents were expressed in thyrocytes by using the rat thyroid-derived FRTL5 cell line.
  • a TSH-stimulated K + current in FRTL5 cells bore the signature linear current- voltage relationship of KCNQl - KCNE2 channels and was inhibited by the KCNQ-specific antagonist XE991 (Fig. 4h,i).
  • KCNQl -KCNE2 channels are expressed in human and rodent thyrocytes, where they generate a TSH-stimulated, constitutively-active K + current.
  • KCNE2 is required for normal thyroid ⁇ accumulation
  • Thyroid hormone requirements are especially high in early development.
  • Kcne2 deletion causes a thyroid ⁇ accumulation defect, which, in turn, causes a thyroid hormone biosynthesis defect, the gross phenotypic effects of which are particularly striking in pups feeding from Kcne2 ⁇ ' ⁇ dams.
  • Kcne2 ⁇ ' ⁇ pups feeding from Kcne2 +I+ dams had higher stomach and thyroid 124 I counts (measured as peak counts per ml), and higher thyroid to stomach count ratios, than did Kcne2 +I+ pups feeding from Kcne2 ⁇ ' ⁇ dams (Fig. 6a-c). This suggested that the surrogating dams' genotype was crucial in determining thyroid 124 I uptake of pups.
  • pup genotype also had a notable effect, because when pups of either genotype were fed from Kcne2 +I+ dams, Kcne2 +I+ pups still had an almost twofold higher thyroid to stomach count ratio at 48-72 h compared to Kcne2 ⁇ ' ⁇ pups (Figs. 5f and Fig. 6c).
  • RAIU thyroid radioactive iodide uptake
  • Kcne2 +I+ and Kcne2 ⁇ ' ⁇ pups showed no significant dam-genotype-independent differences in their feeding rates, as measured by weight gain (Fig. 6e and Supplementary Fig. 3). Furthermore, pups were latched on to dams of either genotype for the entire period under study (30 or 60 min). Thus, the milk ejection defects of Kcne2 ⁇ ' ⁇ dams were not related to behavioral differences in either pups or dams. Hypothyroid rats have previously been shown to have impaired milk ejection owing to reduced serum oxytocin compared to euthyroid rats 27 .
  • KCNQ1 messenger RNA was found to be expressed at a higher level in human thyroid than in the heart or stomach 29 , but its role in the thyroid has not previously been reported.
  • Kcnql gene-disrupted mice like the Kcne2 ⁇ ' ⁇ mice described here, were previously found to have enlarged hearts and thickened ventricular walls, but the mechanistic basis for this was not described 30 ' 31 .
  • T 3 and T 4 biosynthesis requires active ⁇ transport in the thyroid, where ⁇ concentrations reach 20-40 times that of the plasma.
  • NIS located on the basolateral side of the thyrocytes, which are thyroid epithelial cells that encircle the colloid, transports ⁇ into the thyrocyte; at the cell-colloid interface, ⁇ ion is oxidized and covalently incorporated into thyroglobulin for thyroid hormone production 17 .
  • NIS function requires a basolateral Na + /K + ATPase for Na + efflux, but the necessity for other channels or transporters in this process is not known.
  • KCNQ1-KCNE2 as a TSH- stimulated thyrocyte K + channel crucial for normal thyroid ⁇ accumulation and probably expressed predominantly at the basolateral membrane.
  • Kcne2 ⁇ ' ⁇ pups are less efficient at accumulating thyroid ⁇ compared to Kcne2 +I+ pups when both are fed by Kcne2 +I+ dams, but have similar ability to accumulate thyroid ⁇ when fed by Kcne2 ⁇ ' ⁇ dams.
  • a defect in thyroid ⁇ accumulation in Kcne2 ⁇ ' ⁇ pups is partially balanced by other factors, including adaptation to their development in a low maternal T 4 environment in the womb and their being initially fed with poorly ejected, low-T 4 milk. Part of this adaptation may involve reduced ⁇ excretion by Kcne2 ⁇ ' ⁇ pups, consistent with previous reports showing reduced ⁇ excretion in hypothyroidism .
  • the phenotypes described here for Kcne2 ⁇ ' ⁇ pups bred from homozygous Kcne2 ⁇ ' ⁇ crosses include features, such as alopecia and cardiac hypertrophy, not always observed in hypothyroid mouse models 33 . This apparent discrepancy may at least partly be explained by the fact that we studied Kcne2 ⁇ ' ⁇ pups derived from Kcne2 ⁇ ' ⁇ dams, whereas heterozygous crosses are typically used. It may also point to additional pathogenesis caused by Kcne2 deficiency beyond thyroid impairment that is treatable by thyroid hormone supplementation.
  • KCNQl is expressed in both thyroid and mammary gland epithelium; in the mammary gland, KCNQl may assemble with KCNE3 to contribute to K + homeostasis 35 .
  • KCNE2 in mammary epithelial function should not be ruled out, our PET data indicate that mammary gland ⁇ uptake is not impaired in Kcne2 ⁇ ' ⁇ dams.
  • QT interval (QTc) on the electrocardiogram 46 a hallmark of loss-of- function mutations in KCNE2 and KCNQl 2,6 , and with atrial fibrillation, an increasingly prevalent disease in the aging population 47 ' 48 that is also associated with some KCNQl and KCNE2 gene variants 9 ' 12 .
  • QTc QT interval
  • arrhythmogenic owing to primary electrical defects in myocyte K + channels containing these subunits— and also contribute to cardiac structural abnormalities, as a secondary effect of thyroid dysfunction due to defective thyroid KCNQl -KCNE2 channels.
  • Another step in the method of the present invention is observing whether the genome of the patient contains at least one copy of KCNQl or KCNE2 allele having a genetic alteration.
  • the determination whether or not there is a genetic alteration may be carried out by the medical practitioner who is examining the patient, or by a third party. For example, the determination can be carried out by a laboratory technician in a laboratory that specializes in identifying genetic alterations. The laboratory then informs the medical practitioner of the results by, for example, providing the medical practitioner with a written or oral report. In such a case, the medical practitioner observes whether the genome of the patient contains at least one copy of a KCNQl or KCNE2 allele having a genetic alteration by reading the report.
  • the genome of a patient generally contains two each of the KCNQl and
  • KCNE2 alleles are any of one or more alternative forms of a gene. In an organism, two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.
  • genetic alteration refers to any changes in one or more of the nucleic acid molecules in the nucleotide coding sequence of wild-type KCNQl or KCNE2 that leads to a change in the amino acid sequence of wild-type KCNQl or KCNE2.
  • a KCNQl or KCNE2 allele that has a nucleotide coding sequence that leads to a change in the amino acid sequence different from the wild-type KCNQl or KCNE2 constitutes one or more genetic alterations.
  • genetic alterations include one or more nucleotide additions, deletions, substitutions, etc, and combinations thereof. The genetic variation may, or may not, result in a frame shift.
  • the genetic alteration can occur at any nucleotide position(s) in the nucleotide sequence of KCNQl or KCNE2.
  • the genetic alteration can occur at the beginning, middle or end of the nucleotide sequence.
  • Nucleotide additions and deletions refer to the addition and deletion, respectively, of one or more nucleotides in the nucleotide sequence of wild-type KCNQ 1 or KCNE2. If more than one nucleotide is added or deleted, the additions and deletions can be contiguous or non-contiguous. Any nucleotide (A, T, C, G), and any combination thereof, can be added or deleted. Additions and deletions may result in a frame shift, or may not result in a frame shift.
  • a nucleotide substitution refers to the replacement of a nucleotide with a different nucleotide.
  • An example of a substitution is a single nucleotide polymorphism.
  • a single nucleotide addition, deletion, or substitution within the genome of a person is a genetic alteration, which is herein referred to as a single nucleotide polymorphism (SNP). More specifically, a SNP may be a single base insertion or deletion variant.
  • a SNP substitution can be considered a transition or a transversion.
  • a transition is the replacement of one purine nucleotide by another purine nucleotide, or one pyrimidine by another pyrimidine.
  • a transversion is the replacement of a purine by a pyrimidine, or vice versa.
  • Mutations to KCNQl or KCNE2 are known in the art and are associated with cardiac and neurological diseases. Generally these diseases are caused by gain of function mutations or loss of function mutations, the former allowing more potassium to pass than a wild type allele allows; the latter allowing less.
  • a genetic alteration may occur within one copy or both copies of a
  • a patient's homologous chromosomes may comprise identical alleles of the KCNQl or KCNE2 gene at corresponding loci, in which case, the patient's KCNQl or KCNE2 genotype is homozygous for the KCNQl or KCNE2 gene.
  • a patient's homologous chromosomes may not comprise identical alleles of the KCNQl or KCNE2 gene at corresponding loci, in which case, the person's KCNQl or KCNE2 genotype is heterozygous for the KCNQl OR KCNE2 gene.
  • the patient's KCNQl or KCNE2 genotype can be homozygous or heterozygous for any genetic alteration, such as those mentioned above.
  • a patient may be homozygous or heterozygous for any SNP.
  • the determination of a genetic alteration comprises observing expression of a KCNQ 1 or KCNE2 protein containing an amino acid alteration.
  • amino acid alteration refers to any changes in the amino acid sequence of wild-type KCNQl or KCNE2 protein.
  • KCNQl or KCNE2 proteins that contain an amino acid alteration will have a different amino acid sequence than wild- type KCNQl or KCNE2 protein.
  • amino acid alterations include one or more amino acid additions, deletions, substitutions, etc. and combinations thereof, e.g. any of the amino acid alterations caused by the genetic alterations described above.
  • non-synonymous codon change An amino acid substitution that changes a codon coding for one amino acid to a codon coding for a different amino acid is referred to as a non-synonymous codon change, or missense mutation.
  • a non-synonymous codon change is a nonsense mutation, which results in the formation of a stop codon, thereby leading to premature termination of a polypeptide chain and a defective protein.
  • correlate or “correlating” refers to relating the presence of a
  • KCNQ1 or KCNE2 allele having a genetic alteration with creates susceptibility to or causes thyroid disease The determination whether a KCNQ1 or KCNE2 allele has a genetic alteration can be carried out without the need for a qualified medical practitioner. For example, a technician in a laboratory that specializes in identifying genetic alterations can perform the correlation step, and inform the medical practitioner of the results.
  • a sample containing the patient's DNA is obtained.
  • samples include blood, salvia, urine and epithelial cells.
  • the sample can be obtained by any method known to those in the art.
  • Suitable methods include, for example, venous puncture of a vein to obtain a blood sample and cheek cell scraping to obtain a buccal sample.
  • DNA can be isolated from the sample by any method known to those in the art.
  • commercial kits such as the QIAGEN System (QIAmp DNA Blood Midi Kit, Hilder, Germany) can be used to isolate DNA.
  • the DNA is optionally amplified by methods known in the art.
  • One suitable method is the polymerase chain reaction (PCR) method described by Saiki et al., Science 239:487 (1988), U.S. Patent No. 4,683,195 and Sambrook et al. (Eds.),
  • oligonucleotide primers complementary to a nucleotide sequence flanking and/or present at the site of the genetic alteration of the allele can be used to amplify the allele.
  • the isolated DNA is used to determine whether an allele containing a genetic alteration is present in the sample.
  • the presence of an allele containing a genetic alteration can be determined by any method known to those skilled in the art.
  • One method is to sequence the isolated DNA and compare the sequence to that of wild-type KCNQ1 or KCNE2.
  • nucleic acid probes and polymerase chain reaction (PCR).
  • Methods for making and using nucleic acid probes are well documented in the art. For example, see Keller GH and Manak MM, DNA Probes, 2nd ed., Macmillan Publishers Ltd., England (1991) and Hames BD and Higgins SJ, eds., Gene Probes I and Gene Probes II, IRL Press, Oxford (1995).
  • oligonucleotides containing either the wild-type or an allele containing a genetic alteration are hybridized under stringent conditions to dried agarose gels containing target RNA or DNA digested with an appropriate restriction
  • oligonucleotide probe hybridizes to the target DNA or RNA detectably better when the probe and the target are perfectly complementary.
  • oligonucleotide probes for a wild-type and an allele containing a genetic alteration being assayed are prepared.
  • Each oligonucleotide probe is complementary to a sequence that straddles the nucleotides at the site of the genetic alteration. Thus, a gap is created between the two hybridized probes.
  • the gap is filled with a mixture of a polymerase, a ligase, and the nucleotide complementary to that at the position to form a ligated oligonucleotide product.
  • a polymerase a polymerase
  • a ligase a ligase
  • the nucleotide complementary to that at the position to form a ligated oligonucleotide product Either of the oligonucleotides or the nucleotide filling the gap may be labelled by methods known in the art.
  • the ligated oligonucleotide product can be amplified by denaturing it from the target, hybridizing it to additional oligonucleotide complement pairs, and filling the gap again, this time with the complement of the nucleotide that filled the gap in the first step.
  • the oligonucleotide product can be separated by size and the label is detected by methods known in the art.
  • Alleles containing a genetic alteration may also be detected if they create or abolish restriction sites; see Baker et al, Science 244, 217-221 (1989).
  • Some additional examples of the use of restriction analysis to assay point mutations are given in Weinberg et al, U.S. Patent 4,786,718 and Sands, M.S. and Birkenmeier, E.H., Proc. Natl. Acad. Sci. USA 90:6567-6571 (1993).
  • PCR-SSCP polymerase chain reaction products
  • a sample containing protein is obtained.
  • the sample can be any sample which contains protein. Examples of such samples include blood and spinal fluid.
  • the sample can be obtained by any method known to those in the art.
  • Protein can be isolated from the sample by any method known to those in the art.
  • commercial kits such as the Mono Q ion exchange
  • the protein can be used, for example, to generate antibodies.
  • the antibody may be polyclonal or monoclonal.
  • Polyclonal antibodies can be isolated from mammals that have been inoculated with the protein in accordance with methods known in the art.
  • polyclonal antibodies may be produced by injecting a host mammal, such as a rabbit, mouse, rat, or goat, with the protein or fragment thereof capable of producing antibodies that distinguish between proteins containing amino acid alterations and wild-type protein.
  • the peptide or peptide fragment injected may contain the wild-type sequence or the sequence containing the amino acid alteration.
  • Sera from the mammal are extracted and screened to obtain polyclonal antibodies that are specific to the peptide or peptide fragment.
  • the antibodies are preferably monoclonal.
  • Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in Nature 256, 495-497 (1975) and by
  • a host mammal is inoculated with a peptide or peptide fragment as described above, and then boosted. Spleens are collected from inoculated mammals a few days after the final boost. Cell suspensions from the spleens are fused with a tumor cell in accordance with the general method described by Kohler and Milstein in Nature 256, 495-497 (1975). See also Campbell, "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burdon et al., Eds, Laboratoty Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985).
  • a peptide fragment must contain sufficient amino acid residues to define the epitope of the molecule being detected (e.g., distinguish between wild-type protein and proteins containing amino acid alterations).
  • the antibodies can, for example, be used to observe the presence of KCNQ1 or KCNE2 proteins containing amino acid alterations. Suitable methods include, for example, a western blot and an ELISA assay.
  • hyperthyroidism Too much thyroid hormone from an overactive thyroid gland is called hyperthyroidism, because it speeds up the body's metabolism.
  • Hyperthyroidism occurs in about 1 percent of all women, who get this condition more often than men.
  • One of the most frequent forms of hyperthyroidism is known as Graves' disease
  • Hyperthyroidism the result of an overactive thyroid, more commonly affects women between the ages of 20 and 40, but men can also develop this condition.
  • Symptoms can include: Muscle weakness; Trembling hands; Rapid heartbeat; Fatigue; Weight loss; Diarrhea or frequent bowel movements; Irritability and anxiety; Vision problems (irritated eyes or difficulty seeing);. Menstrual irregularities; Intolerance to heat and increased sweating; Infertility.
  • Graves' disease is the most common cause of hyperthyroidism. It occurs when the immune system produces antibodies that attack the thyroid gland, making it produce too many thyroid hormones and creating a hormone imbalance. This condition happens often in people with a family history of thyroid disease. In some patients with Graves' disease, one of the noticeable symptoms may be swelling behind the eyes, causing discomfort or increased tearing or causing the eyes to push forward or bulge.
  • Inflammation irritation and swelling
  • Non-cancerous growths of the thyroid gland or pituitary gland Taking large amounts of thyroid hormone
  • Mutations that cause gain of function in KCNQ1 or KCNE2 can cause, or create susceptibility to, hyperthyroidism.
  • hypothyroidism Too little thyroid hormone from an underactive thyroid gland is called hypothyroidism, another hormone imbalance caused by thyroid problems.
  • hypothyroidism the body's metabolism is slowed.
  • TSH thyroid-stimulating hormone
  • the problem is caused by the thyroid conditions or by the pituitary gland, the result is that the thyroid is underproducing hormones, causing many physical and mental processes to become sluggish.
  • the body consumes less oxygen and produces less body heat.
  • hypothyroidism which occurs when an underactive thyroid does not produce enough hormones, can be a dangerous condition if untreated. Instead of the bodily systems speeding up and overheating, they slow down in a variety of ways.
  • This thyroid disease's symptoms include the following: Fatigue; Mental depression; Sluggishness; Feeling cold; Weight gain; Dry skin and hair; Constipation; Menstrual irregularities [000107]
  • myxedema The most severe expression of hypothyroidism may be referred to as myxedema. If you have severe hypothyroidism, a significant injury, infection, or exposure to cold or certain medications may trigger a life-threatening condition called myxedema coma. This condition may cause a patient to lose consciousness and to develop hypothermia, a life-threatening low body temperature
  • hypothyroidism inflammation of the thyroid gland, which damages the gland's cells. Autoimmune or Hashimoto's thyroiditis, in which the immune system attacks the thyroid gland, is the most common example of this. Some women develop hypothyroidism after pregancy (often referred to as "postpartum throiditis").
  • hypothyroidism Other common causes of hypothyroidism include: Congenital (birth) defects; Radiation treatments to the neck to treat different cancers, which may also damage the thyroid glands; Radioactive iodine used to treat an overactive thyroid (hyperthyroidism); Viral thyroiditis, which may case hyperthyroidism and is often followed by temporary or permanent hypothyroidism; Certain drugs; and Sheehan syndrome, a condition that may occur in a woman who bleeds severely during pregnancy or childbirth and causes destruction of the pituitary gland.
  • KCNQ 1 or KCNE2 Compounds that inhibit the activity of KCNQ 1 or KCNE2; that promote the activity of KCNQ 1 or KCNE2, or that open KCNQ1-KCNE2 channels, and that block KCNQ1-KCNE2 channels, are known in the art. See for example and without limitation, Xiong Q, et al (2008), which is incorporated herein in its entirety. Such known compounds may be repurposed to treat thryoid diseases, as described herein.
  • Non-human animals includes all vertebrates, e.g., mammals and non- mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows and horses.
  • the subject may also be livestock such as, cattle, swine, sheep, poultry, and horses, or pets, such as dogs and cats.
  • the subject may be male or female, and may be elderly, an adult, adolescent, child, or infant.
  • the human subject may be Caucasian, of priman, asian, Semitic, or of other or mixed racial background.
  • Preferred subjects include human patients suffering from or at risk for thyroid disease.
  • thyroid hormone administration and surrogacy For thyroid hormone administration, we injected pups intraperitoneally every other day with 130 ng T 4 and 13 ng T 3 per g body weight, beginning at postnatal day 1 ; we anesthetized those less than 1 week old in an ice water bath for 10-20 s before injection. We injected virgin adults intraperitoneally every other day with 1 ⁇ g per g body weight T 3 and 0.4 ⁇ g per g body weight T 4 for 2 weeks. We fed pregnant dams T 4 ad libitum at 5 mg ⁇ 1 in their drinking water, from the last 2 weeks of gestation to the weaning of their pups, with dosage and preparation as previously described 55 .
  • Imaging was performed on a Concorde Microsystems R4 microPET Scanner (Siemens), with 24 detector modules providing 7.9 cm axial and 12 cm transaxial field of view. Acquisitions were performed in three-dimensional list mode to permit either dynamic or static reconstruction. A reconstructed FWHM resolution of 1.9 mm is achievable in the center of the axial field of view.
  • Masson's Trichrome staining of cardiac and hepatic sections was performed to detect collagen deposition.
  • Goat polyclonal anti-KCNQl (pan-species) primary antibody (Santa Cruz Biotech #SC- 10646) was used at lmg/ml; in-house rabbit polyclonal, site-directed, anti-KCNE2 (pan- species) serum was diluted 1 :5000 after column-enriching IgG; and in-house affinity-purified, site-directed, rabbit polyclonal anti-NIS antibodies (one raised against a rat NIS epitope for mouse thyroid slides, and one raised against a human NIS epitope for human thyroid slides) were used at 1 ⁇ g/ml.
  • the tissue sections were blocked for 30 min in 10% normal goat, mouse or rabbit serum, 2% BSA in PBS, followed by 8 min Avidin/Biotin block.
  • the primary antibody incubation (3 hr) was followed by incubation with biotinylated anti-rabbit or goat IgG as appropriate (ABC kit from Vector labs).
  • the secondary detection was performed with Streptavidin-HRP D (Ventana Medical Systems), followed by incubation with Tyramide-Alexa Fluor secondary antibodies (Invitrogen). Immunostained slides were viewed with a Zeiss Axiovert 200 widefield microscope and pictures were acquired using MetaMorph 7.1 software (Molecular Devices).
  • Membranes were incubated for 1 min with the SuperSignal ECL reagent (Pierce) then exposed on BioMax Light Film (Kodak) and developed using an RP X-OMAT Processor (Kodak). Membrane preparations from FRTL5 cells were processed similarly. FRTL5 cells were grown for 6 days in the absence or presence of TSH, or incubated for 12 hours with/without cAMP (the major
  • FRTL5 cells were grown in TSH (+TSH) or starved of TSH in culture for 6 days (-TSH). Alternatively, FRTL5 cells were incubated without/with cAMP for 12 hours.
  • Echocardiography Transthoracic echocardiograms were recorded in 3- week-old conscious-sedated (1% isoflurane in 100% oxygen) mice with a Sequoia C256 and 15L8 probe (Acuson, Mountain View, CA, USA).
  • Left ventricular end-systolic dimension (LVESD), left ventricular end-diastolic dimension (LVEDD), interventricular septal thickness (IVST), anterior wall (AW) and posterior wall (PW) thickness both in diastole and systole, were measured at the level of the papillary muscles on the short-axis view using 2-dimensional guided M-mode imaging at 3 cardiac cycles.
  • immunization leads to fetal loss in specific allogeneic pregnancies.

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Abstract

L'invention concerne un procédé permettant de traiter la maladie hyperthyrodienne par administration au patient concerné un composé que l'on sait capable de bloquer l'activité de KCNQl ou KCNE2 ou qui bloque le canal KCNQ1-KCNE2
PCT/US2010/049488 2009-09-18 2010-09-20 Kcnq1 et kcne2 dans la maladie thyrodienne WO2011035239A1 (fr)

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US9566333B2 (en) 2011-12-19 2017-02-14 Hill's Pet Nutrition, Inc. Compositions and methods for diagnosing and treating hyperthyroidism in companion animals
WO2018002147A1 (fr) * 2016-06-30 2018-01-04 INSERM (Institut National de la Santé et de la Recherche Médicale) Détection de mutations de boréaline pour le diagnostic de la dysgénésie de la thyroïde

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US20090220949A1 (en) * 2005-06-07 2009-09-03 Fondzione Salvatore Maugeri Clinica Del Lavoro E Della Riabilitazione I.R.C.C.S. Mutations Associated with the Long QT Syndrome and Diagnostic Use Thereof

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US20030032786A1 (en) * 2001-01-24 2003-02-13 Han Chang Polynucleotide encoding a novel human potassium channel beta-subunit, K+betaM2
US20030162192A1 (en) * 2001-08-20 2003-08-28 Sotos John G. Polymorphisms associated with ion-channel disease
US20090220949A1 (en) * 2005-06-07 2009-09-03 Fondzione Salvatore Maugeri Clinica Del Lavoro E Della Riabilitazione I.R.C.C.S. Mutations Associated with the Long QT Syndrome and Diagnostic Use Thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9566333B2 (en) 2011-12-19 2017-02-14 Hill's Pet Nutrition, Inc. Compositions and methods for diagnosing and treating hyperthyroidism in companion animals
RU2612901C2 (ru) * 2011-12-19 2017-03-13 Хилл'С Пет Ньютришн, Инк. Композиции и способы диагностики и лечения гипертиреоза у животных-компаньонов
WO2018002147A1 (fr) * 2016-06-30 2018-01-04 INSERM (Institut National de la Santé et de la Recherche Médicale) Détection de mutations de boréaline pour le diagnostic de la dysgénésie de la thyroïde

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