WO2011035239A1 - Kcnq1 and kcne2 in thyroid disease - Google Patents

Kcnq1 and kcne2 in thyroid disease 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

The invention relates to a method to treat hyperthyroid disease by administering to a subject in need thereof a compound known to block the activity of KCNQl or KCNE2 or that blocks the KCNQ1-KCNE2 channel.

Description

KCNQl AND KCNE2 IN THYROID DISEASE
[0001] This invention was made with Government support from the American
Heart Association under Award No. 0855756D. The Government has certain rights in this invention.
Cross-Reference to Related Application
[0002] This application claims the benefit of U.S. Provisional Application No.
61/243,938, filed September 18, 2009, which is incorporated herein by reference in its entirety.
Background of the Invention
[0003] 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 (IKs) by assembling with the KCNEl (originally named MinK) single-transmembrane domain β subunit1 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 properties5 7. Inherited mutations in the genes encoding KCNQl, hERG, KCNEl and KCNE2 are all associated with life-threatening cardiac arrhythmias, including long QT syndrome3'4'6'8, and may have a role in atrial fibrillation9 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.
[0004] 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 cells13, 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 hyperplasia14'15.
[0005] 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 T3 and T4 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 T3 and T4, iodide (Γ) deficiency disorders are still prevalent in many areas of the world and are thus at the forefront of global health initiatives. Γ enters thyrocytes via the basolaterally located Na+/F symporter (NIS)16'17 and exits apically into the colloid, where it is covalently incorporated into thyroglobulin, the precursor of T3 and T4. NIS-mediated Γ 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.
[0006] In this invention, it is shown that potassium channel subunits KCNQ1 and
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.
Summary of the Invention
[0007] Thyroid dysfunction is a global health concern, causing defects including neurodevelopmental disorders, dwarfism and cardiac arrhythmia. Here, we show that 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
tetraiodothyronine (T4) content and halved litter size. Kcne2-deficient 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 (T3) and T4 administration to pups, by supplementing dams with T4 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. These data provide a possible new therapeutic target for thyroid disorders and raise the possibility of an endocrine component to previously identified KCNE2- and KCNQ1 -linked human cardiac arrhythmias. Brief Description of the Drawings
[0008] Figure 1 Kcne2 disruption causes cardiac hypertrophy, fibrosis and reduced fractional shortening, (a) Left, external view of representative hearts from 3- week-old Kcne2+I+ (left) and Kcne2~'~ (right) mice from homozygous crosses. Right, mean heart mass, body mass and heart weight to body weight ratio (HW/BW; mg per g) for mice as in left (n = 9-14 per group). * < 0.000001. Error bars indicate s.e.m. (b) Representative transthoracic echocardiograms for a 3 -week-old Kcne2+I+ pup from a Kcne2+I+ dam and a Kcne2~'~ pup from a Kcne2~'~ dam. (c) Mean echocardiographic parameters from recordings as in b. d or s, diastolic or systolic; LVAW, left ventricular anterior wall thickness; LVID, left ventricular internal diameter; LVPW, left ventricular posterior wall thickness; n = 4 per group. Error bars indicate s.e.m. * < 0.05; **P < 0.005; ***P < 0.0005, by one-way analysis of variance (ANOVA). (d) Left, mean cell capacitance for ventricular myocytes isolated from 3 -week-old pups from homozygous crosses, n = 6-8 per group; *P < 0.001. Error bars indicate s.e.m. Right, representative whole-cell recordings from ventricular myocytes. Insets, voltage protocol and scale bars, (e) Mean peak current densities for Kcne2+I+ (blue) and Kcne2~'~ (red) myocytes as in d, n = 6-8 per group; * < 0.05. Error bars indicate s.e.m. (f) Left, current inactivation τ values for Kcne2+I+ (blue) versus Kcne2~'~ (red) myocytes as in e. Right, current amplitudes from double exponential fits for Kcne2+I+ (solid) versus Kcne2~'~ (open) myocytes as in e, n = 6-8 per group. * < 0.001. Error bars indicate s.e.m. Current decay was best fit with two exponentials with parameters resembling Ito,f and IK,S1OW and a steady-state current, Iss. (g-i) Cardiac tissue from 3-week-old Kcne2~'~ mice bred from Kcne2~'~ dams, (g) Representative necropsy, (h) Left, representative Masson's trichrome- stained left ventricle showing collagen (blue), indicative of fibrosis. Scale bar, 150 μιη. Right, representative H&E-stained papillary muscle. Scale bar, 30 μιη. (i) Representative Masson's trichrome-stained liver, showing marked perisinusoidal fibrosis (blue). Scale bar, 200 μιη. (j) Cardiac tissue from 15-month-old Kcne2~'~ mice bred from Kcne2+I~ dams; similar results were observed in four of four mice evaluated. Left, representative necropsy showing cardiomegaly; middle and right, representative Masson's trichrome- stained left ventricle, showing fibrosis (blue). Scale bars: middle, 100 μιη; right, 30 μιη. [0009] Figure 2 Kcne2 disruption causes embryonic lethality, dwarfism and alopecia, (a) Left, mean live births per litter for wild-type and Tcne2-disrupted crosses as indicated; n = 19-50 litters per group. * < 0.01. Error bars indicate s.e.m. Right, genotype of surviving pups (%). Numbers of mice are shown in parentheses, (b)
Representative 3 -week-old pups from Kcne2~'~ x Kcne2~'~ and K n 2 x K n 2 crosses, (c) Representative X-ray images of 5 -week-old pups from Kcne2~'~ x Kcne2~'~ and Kcne2+I+ x Kcne2+I+ crosses, (d) Mean body mass at 3-6 weeks of age for pups from Kcne2+I+ and Tcne2-disrupted crosses as indicated; n = 15-50 pups per group. *P < 0.05. Error bars indicate s.e.m. (e) Representative 5-week-old Kcne2+I+ and Kcne2~'~ mice from homozygous crosses, (f) Representative close-up views of skin from mice such as those shown in e. (g) Representative micrograph of H&E-stained dermis sections from a Kcne2~'~ mouse such as those shown in e. Scale bar, 300 μιη. (h) Representative 1 -year- old K n 2 and Kcne2~'~ mice from Kcne2+I~ x Kcne2+I~ crosses, (i) Representative micrograph of H&E-stained dermis sections from the Kcne2~'~ mouse shown in h. Inset, detail from the boxed region, showing the border zone between normal hair and alopecia, with abrupt cessation of mature hair follicles. Scale bar, 600 μιη. (j) H&E-stained sections showing hair follicles in dermis from Kcne2+I+ and Kcne2~'~ mice such as those shown in i. Scale bars, 20 μιη.
[00010] Figure 3 Kcne2~'~ mice are hypothyroid and treatable with T3 and T4 or wild-type surrogacy, (a) Left, serum T4 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. (b) Mean mass of thyroid glands from 12-month-old Kcne2+I+, Kcne2+I~ and Kcne2~'~ mice from Kcne2+I~ x Kcne2+I~ crosses, weighed postmortem. Numbers of mice are shown in parentheses. *P < 0.005; **P < 1 x 10~4; ***P < 1 x 10~9. Error bars indicate s.e.m. (c) Serum T4 in pregnant Kcne2+I+ and Kcne2~'~ mice. Numbers of mice are shown in parentheses. *P < 0.001 compared to Kcne2+I+ . Error bars indicate s.e.m. (d) Representative 3 -week-old pups from homozygous crosses surrogated (Sgt) with dams of opposite genotype, (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 T3 and T4] injection (P) or by T4 supplementation of their mothers (D); n = 9-23 pups per group. Error bars indicate s.e.m. Untreated groups (/) from Figure 2d are shown for comparison, (f) Representative 12-week-old Kcne2~'~ mouse before (left) and after (right) 10 d T3 and T4 administration every other day. Results were consistent in five of five mice evaluated, (g) Left, histology of the skin of the mouse in f after T3 and T4 treatment, showing recovery of normal hair follicles. Scale bar, 200 μιη. Right, percentage of mice with normal hair growth grouped according to parental genotype. Groups were untreated, surrogated with dams (genotype as indicated) or treated directly by T3 and T4 injection every other day (P) or by T4 supplementation of their mothers; n = 16-23 mice per group, (h) Representative necropsies of 3 -week-old Kcne2+I+ and Kcne2~'~ mice bred from homozygous crosses, with or without surrogacy with mothers of the opposite genotype, (i) Mean heart weight to body weight ratio (HW/BW; mg per g) for mice from the same experimental groups as in h, n = 7-9 per group. *P < 0.05 compared to values for nonsurrogated pups (taken from Fig. lc and the key in Fig. 3j). Error bars indicate s.e.m. (j) Mean
echocardiographic parameters for 3 -week-old Kcne2+I+ and Kcne2~'~ mice bred from homozygous crosses, with or without surrogacy with mothers of the opposite genotype, n = 5 mice per group. * < 0.05 compared to values for nonsurrogated pups (taken from Fig. lc). Error bars indicate s.e.m.
[00011] Figure 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 μιη. Representative of n = 2 sections, (b) Western immunoblots (IB) using antibodies raised against KCNE2 (arrows indicate three bands presumed to correspond to the three glycosylation states of KCNE2) and KCNQ1 (bottom arrow, monomer; top arrow, multimer) in thyroid tissue from K n 2 and Kcne2~'~ mice (from Kcne2+I~ x Kcne2+I~ crosses). Representative of n = 3 experiments, (c) Immunofluorescence using antibodies raised against KCNE2, KCNQ1 and NIS in sections of thyrocytes from 3-month-old K n 2 mice (from Kcne2+I~ x Kcne2+I~ crosses). Asterisks indicate colloid. Scale bars, 4 μιη. Representative of n = 3 thyroids, (d) Immunofluorescence using antibodies raised against KCNE2 and KCNQ1 in sections of thyrocytes from 3-month-old Kcne2~'~ mice (from Kcne2+I~ x Kcne2+I~ crosses). Asterisk indicates colloid. Scale bars, 4 μιη. Representative of n = 2 thyroids, (e) H&E staining of sections from adult Kcne2 and Kcne2~'~ thyroid glands (from Kcne2+I~ x Kcne2+I~ crosses). Scale bars, 20 μιη.
Representative of 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 μιη. Representative of n = 2 thyroids per genotype, (g) Western
immunoblots (IB) of membrane fractions from FRTL5 cells with and without 10 h of incubation with cyclic AMP, using antibodies raised against KCNE2 (arrows indicate the three glycosylation states) and from FRTL5 cells with and without 6 d of TSH incubation, using antibodies raised against KCNQl (bottom arrow, monomer; top arrow, tetramer). Representative of n = 3 experiments, (h) Representative whole-cell patch- clamp recordings of XE991 -sensitive currents (calculated by digital subtraction of post- XE991 application current from the pre-XE991 application current from the same cell) in FRTL5 cells with or without TSH incubation. Top left inset, voltage protocol. Zero current level is indicated by the dashed line, (i) Mean whole-cell current-voltage relationships for total current (n = 9-16 per group) and XE991 -sensitive current
(calculated by digital subtraction of post-XE991 application current from the pre-XE991 application current from the same cell) (n = 8 or 9 per group) in FRTL5 cells with or without TSH incubation. * < 0.05. Error bars indicate s.e.m
[00012] Figure 5 Kcne2 is required for normal thyroid Γ accumulation, (a)
Representative micro PET images of lactating Kcne2+I+ and Kcne2~'~ dams recorded during the first hour after tail vein injection of 124I. t, thyroid; m, mammary gland. Red indicates highest intensity, as shown by the color intensity scale. Scale bar, 5 mm. (b) Mean 124I accumulation in thyroid relative to mammary gland from imaging as in a, measured as the ratio of maximum radioactivity in each tissue minus mean background count in each mouse, n = 4 mice per group; error bars indicate s.e.m. *P < 1 x 10~8. (c) Mean 124I accumulation in thyroid relative to mammary gland as in a but from 1-72 h after tail vein injection of 124I. n = 4 or 5 mice per group; error bars indicate s.e.m. *P < 0.05. (d) Representative microPET images of preweaning Kcne2+I+ and Kcne2~'~ pups recorded 24-72 h after their mothers (same dams as in a-c) received tail vein injections of 1241. 1, thyroid; s, stomach. Intensity scale as in a. Scale bar, 5 mm. (e) Mean 124 accumulation (in μθ) 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. (f) Mean 124I accumulation in thyroid relative to stomach, measured as the ratio of maximum radioactivity in each tissue minus mean background count in each mouse. The imaging method and pups are as in e. n = 7-12 pups per time point per group; error bars indicate s.e.m. *P < 0.0005.
[00013] Figure 6 Kcne2~'~ dams have a milk ejection defect and produce low-T4 milk, (a) Representative microPET images of preweaning Kcne2+I+ and Kcne2~'~ pups recorded 24-96 h after their surrogate mothers of opposite genotype received tail vein injections of 124I. An intensity scale is shown on the right. Scale bar, 10 mm. (b) Mean peak 124I accumulation (measured as peak μθ mP1) in thyroid and stomach from imaging as in a, measured as 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; **P < 0.01. (c) Mean peak 124I accumulation in thyroid relative to stomach, measured as the ratio of maximum radioactivity in each tissue minus mean background count in each mouse. The imaging method and pups are as in a. **P < 0.001. (d) Effects of pup and dam genotype on pup total thyroid and body 124I accumulation (left), and thyroid RAIU (radioactive iodide uptake as a percentage of total body radioiodine) (right), determined using three-dimensional regions of interest from PET analyses of pup 124I accumulation (surrogated and nonsurrogated) after tail vein injection of dams. n = l- 12 per group. *P < 0.05; **P < 0.005; ***P < 0.0005. Error bars indicate s.e.m. (e) Milk ejection assay. Mean mass of pups before (0 min) and after 30 min feeding period; n = 8- 20; * < 0.01. Oxy' indicates dams injected with oxytocin 10 min before feeding period, (f) Milk T4 concentration, n = 5-7 per group; * < 0.05. (g) Plasma Γ concentration in 3- week-old pups, n = 6 per genotype; error bars indicate s.e.m.
Detailed Description of the Invention
[00014] In the following description, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments which may be practiced. These embodiments are described in detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural, logical and electrical changes may be made without departing from the scope of the present invention. The following description of example embodiments is, therefore, not to be taken in a limited sense, and the scope of the present invention is defined by the appended claims.
[00015] The Abstract is provided to comply with 37 C.F.R. § 1.72(b) to allow the reader to quickly ascertain the nature and gist of the technical disclosure. The Abstract is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.
[00016] Kcne2~'~ pups from Kcne2~'~ dams have cardiomegaly
[00017] Mutations and polymorphisms in human KCNQ1 and KCNE2 are associated with ventricular and atrial cardiac arrhythmias, presumed to arise from dysfunction of the K+ channels they form in cardiac myocytes4'6'18. We previously found that at 3 months of age, Kcne2~'~ mice from Kcne2+I~ x Kcne2+I~ crosses have normal echocardiographic parameters and ventricular myocyte size19. In contrast, here, when we bred Kcne2~'~ pups from Kcne2~'~ x Kcne2~'~ crosses, we found that they showed striking cardiomegaly: more than twofold increased heart mass and more than threefold increased heart weight to body weight ratio, at 3 weeks (Fig. la). Three-week-old Kcne2~'~ pups derived from Kcne2~'~ dams also showed >50% increases in end-diastolic left ventricular internal diameter, left ventricular anterior and posterior wall thickness and a 45% decrease in fractional shortening (Fig. lb,c). The 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 mechanism20 ) to the impaired contractility resulting from sustained hypertrophy.
[00018] 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). The raw amplitudes of two of the predominant mouse ventricular repolarization K+ currents, the slow-activating delayed rectifier K+ current termed IK,S1OW and the fast component of the transient outward K+ current (It0,f), were unaltered by Kcne2 disruption, but, because of the doubling in capacitance, there was a halving in density for each of these currents (Fig. ld-f). In contrast, the raw amplitude of the steady-state K current (Iss) doubled, such that its density did not change with Kcne2 disruption (Fig. ld-f).
[00019] Hearts from 3 -week-old Kcne2~'~ mice from Kcne2~'~ dams (Fig. lg) also showed marked left ventricular fibrosis and papillary muscle degeneration, necrosis and mineralization, features of sustained hypertrophy (Fig. lh). We also observed hepatic fibrosis, suggesting right heart failure (Fig. li), and an unusually pale appearance of the liver (Fig. lg), possibly indicative of hepatic steatosis. Furthermore, although the hearts of 3-month-old Kcne2~'~ mice bred from Kcne2+I~ dams appeared to be normal19, hearts of 1 -year-old Kcne2~'~ mice bred from Kcne2+I~ dams showed cardiomegaly with left ventricular fibrosis (Fig. lj). In sum, it is apparent that parental genotype strongly influences the cardiac phenotype of Kcne2~'~ offspring.
[00020] Kcne2_/~ mice show dwarfism and alopecia
[00021] Aside from their cardiac pathology, 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). Although birth weights were similar in all cases, the mean body weight of pups from Kcne2~'~ x Kcne2~'~ crosses was 40% lower than that of pups from K n 2 x K n 2 crosses at 5 weeks of age (Fig. 2d). Reduced body mass correlated with the maternal Kcne2~'~ genotype, but also to pup genotype, as pups from Kcne2+I~ sires were significantly larger than those from Kcne2~'~ sires (both with Kcne2~'~ dams) (Fig. 2d). By 15 weeks of age, mean body weights were similar regardless of pup genotype
(Supplementary Fig. la).
[00022] Radiological examination revealed retarded skeletal development in the
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).
[00023] Unexpectedly, 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). 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).
[00024] Kcne2~ ~ phenotype is attenuated by TH or Kcne2+/+ milk
[00025] Considering the combination of cardiac hypertrophy, cardiac and hepatic fibrosis, dwarfism, alopecia and skeletal abnormalities indicative of retarded
development in Kcne2~'~ pups, we investigated whether hypothyroidism might be the underlying cause. We first determined serum thyroid hormone concentrations. Indeed, the mean serum T4 concentration in 3 -week-old Kcne2~'~ pups from Kcne2~'~ x Kcne2~'~ crosses was 40% lower, and the mean serum thyroid-stimulating hormone (TSH) concentration twofold greater, than in age-matched Kcne2+I+ pups from Kcne2+I+ x Kcne2+I+ crosses (Fig. 3a), confirming hypothyroidism in the Kcne2~'~ pups. Serum T4 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). By 12-15 months of age, T4 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). Thyroid glands from aged Kcne2~'~ mice bred from Kcne2+I~ x
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 T4 concentration than pregnant Kcne2+I+ dams (Fig. 3c), suggesting an explanation for the exaggerated phenotype and small litter size of Kcne2~'~ pups bred from Kcne2~'~ dams (Fig. 2). Consistent with previous reports for mice and rats (but in contrast to humans)21'22, pregnant mice in our study showed lower serum T4 concentrations than did age- and genotyped-matched nubile adults (Fig. 3c and Supplementary Fig. 2).
[00026] We next tested whether or not switching Kcne2~'~ litters with the litters of
Kcne2+I+ dams (surrogacy) 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. Notably, normal body weight was fully restored in Kcne2~'~ pups from Kcne2~'~ x Kcne2~'~ crosses by surrogacy with K n 2 dams (Fig. 3d,e).
Conversely, surrogacy of Kcne2+I+ pups with Kcne2~'~ dams from birth through to weaning resulted in a mean preweaning body weight similar to that of Kcne2~'~ pups and an intermediate body weight in pups after weaning (Fig. 3d,e). These data suggested the possibility that maternal thyroid hormone passed through milk, perhaps at higher concentrations, or in higher volumes of milk, from wild-type dams, were compensating for the defect in Kcne2~'~ pups. Confirming the role of thyroid hormone in body weight differences, Kcne2~'~ pups born and raised by Kcne2~'~ dams and from Kcne2~'~ sires showed markedly improved body weight by 3 weeks of age after T3 and T4
administration every 48 h from birth (Fig. 3e and Supplementary Fig. lb). Furthermore, T4 supplementation of Kcne2~'~ dams from 2 weeks before birth to weaning also resulted in normal pup body weight (Fig. 3e).
[00027] Dermato logical disorders occur frequently in hypothyroidism23. Notably, we found that alopecia was completely reversed in 5/5 adult Kcne2~'~ mice evaluated, with 2 weeks administration of T3 and T4 every other day (examples are shown in Fig. 3f,g). Alopecia was also completely reversed in 19 of 21 Kcne2~'~ pups after 10 d of T3 and T4 administration every other day, in 19 of 20 Kcne2~'~ pups by surrogacy with Kcne2+I+ dams and in 16 of 16 Kcne2~'~ pups by T4 supplementation of their mothers from 2 weeks before birth to weaning. Conversely, we observed alopecia in 13 of 23 Kcne2+I+ pups surrogated with Kcne2~'~ dams (Fig. 3g).
[00028] Hypothyroidism is associated with dilated and hypertrophic
cardiomyopathies, reduced fractional shortening and heart failure24. Supporting a link between these cardiac defects and the hypothyroidism that we observed in Kcne2~'~ mice, surrogacy of Kcne2~'~ pups with Kcne2+I+ dams resulted in a marked reduction in the relative mass of the heart compared to body mass; conversely, surrogacy of Kcne2+I+ pups with Kcne2~ ~ dams had the opposite effect (Fig. 3 h,i). As observed with nonsurrogated Kcne2~'~ pups (Fig. lg), the liver of Kcne2+I+ pups surrogated with Kcne2~ '~ dams had an unusually pale appearance, possibly indicative of hepatic steatosis (Fig. 3h). Furthermore, echocardiography revealed beneficial effects of surrogacy of Kcne2~'~ pups with Kcne2+I+ dams: in these pups, there was a significant reduction in ventricular wall thickness and chamber diameter and increased fractional shortening compared to nonsurrogated Kcne2~'~ pups (Fig. 3j).
[00029] KCNE2 and KCNQl form a TSH-stimulated thyrocyte K+ channel
[00030] Our data (Figs. 1-3) suggested a potential role for KCNE2 in thyroid hormone biosynthesis. Previous studies have indicated that KCNE2 forms heteromeric channels with the KCNQl K+ channel a subunit in gastric epithelium14'15'25'26. Here we found that both 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). In both human and mouse thyroid, KCNE2 and KCNQl partially co-localized with NIS, the basolateral membrane glycoprotein that mediates active Γ transport, the first step in thyroid hormone biosynthesis. Furthermore, thyroid follicular epithelia in Kcne2~'~ mice showed abnormal architecture; compared to thyrocytes in Kcne2+I+ mice, Kcne2~'~ thyrocytes were often flattened and were less abundant (Fig. 4e,f).
[00031] We next sought to determine whether KCNQl -KCNE2 K+ currents were expressed in thyrocytes by using the rat thyroid-derived FRTL5 cell line. We detected endogenously expressed KCNQl and KCNE2 proteins, whose expression was upregulated by TSH or its major downstream effector, cyclic AMP, in FRTL5 cell membrane fractions (Fig. 4g). We next measured endogenous currents from FRTL5 cells using patch-clamp recording in the whole-cell configuration. 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). In sum, KCNQl -KCNE2 channels are expressed in human and rodent thyrocytes, where they generate a TSH-stimulated, constitutively-active K+ current. [00032] KCNE2 is required for normal thyroid Γ accumulation
[00033] Thyroid hormone requirements are especially high in early development.
Developing fetuses and neonates rely on not only their own thyroid hormone biosynthesis but also maternal T4 in utero and perhaps maternal T4 present in milk. We therefore examined thyroid Γ accumulation, a key step in thyroid hormone biosynthesis, in lactating dams and their pups. We injected 124I only into the tail vein of lactating dams, which we then placed back together with their pups to feed them. We imaged both dams and pups by positron emission tomography (PET). Kcne2~'~ dams showed a striking defect in 124I accumulation in the thyroid, with 75% less accumulation over the first hour after injection and continuing deficiency in the following 3 d, compared to Kcne2+I+ dams (Fig. 5a-c). In pups whose sole source of 124I was dams' milk, Kcne2 deletion caused an 80% reduction in pup thyroid 124I accumulation 24 h after injection of dams, and continuing deficiency for the following 2 d (Fig. 5d,e). When normalized to stomach 124I count, the thyroid 124I count was reduced 87% in Kcne2~'~ pups compared to
Kcne2+I+ pups at 72 h after injection of the dam (Fig. 5f).
[00034] Thus, Kcne2 deletion causes a thyroid Γ 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. To examine the mechanistic basis for this phenotype, and for the beneficial effects of surrogacy by Kcne2+I+ dams, we first performed PET on pups surrogated with dams of opposite genotype. We performed tail vein injections of lactating dams with 124I followed by imaging of the pups feeding from them. Of note, we found that Kcne2~'~ pups feeding from Kcne2+I+ dams had higher stomach and thyroid 124I 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 124I uptake of pups. However, 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).
[00035] We also quantified thyroid radioactive iodide uptake (RAIU), a measure
124 124 of the efficiency of the thyroid at accumulating I from the available total body I, for all surrogated and nonsurrogated pups (Fig. 6d). Total thyroid I counts were higher in both Kcne2+I+ and Kcne2~'~ pups when feeding from Kcne2+I+ dams than when feeding from Kcne2~'~ dams, whereas total body 124I counts were significantly higher only for Kcne2~'~ pups feeding from Kcne2+I+ dams compared to other groups (Fig. 6d). In contrast, thyroid RAIU was significantly higher only in Kcne2+I+ pups feeding from Kcne2+I+ dams compared to other groups (Fig. 6d). These data demonstrate again that Kcne2~'~ pups' thyroids are less efficient than those of Kcne2+I+ pups at accumulating Γ, but also indicate that Kcne2~'~ dams supply less Γ to their pups than do Kcne2+I+ dams. These results also indicate that Kcne2~'~ pups are better at accumulating total body Γ than are Kcne2+I+ pups.
[00036] To examine the poor delivery of Γ from Kcne2~'~ dams, we compared milk ejection from Kcne2+I+ and Kcne2~'~ dams by weighing pups before and after feeding, as previously described27. Kcne2~'~ dams had a highly significant milk ejection defect, manifested as pups (of either genotype) failing to gain weight (from milk ingestion) during the first 30 min of feeding from Kcne2~'~ dams, in sharp contrast to pups feeding from Kcne2+I+ dams (Fig. 6e). We observed similar results over the first 60 min of feeding (Supplementary Fig. 3). Notably, 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 rats27. Accordingly, we found that injection of Kcne2~'~ dams with oxytocin returned their milk ejection to the same level as that of Kcne2+I+ dams (Fig. 6e). The milk ejection defect was probably a dominant factor in the beneficial effects of Kcne2+I+ surrogacy and the negative effects of Kcne2~'~ surrogacy. Additionally, however, milk from Kcne2~'~ dams contained only half as much T4 as that from Kcne2+I+ dams (Fig. 6f), potentially also contributing to the observed effects of surrogacy.
[00037] Finally, to address the superior total body accumulation of Γ by Kcne2~'~ pups, we found equal serum Γ concentrations in nonsurrogated 3 -week-old Kcne2+I+ and Kcne2~ ~ pups (Fig. 6g), suggesting that, despite inferior milk ejection by Kcne2~ ~ dams, Kcne2~'~ pups were able to maintain normal plasma Γ concentrations. This was not unexpected, given that hypothyroidism is known to result in decreased Γ excretion28.
[00038] More than a decade ago, KCNQ1 messenger RNA was found to be expressed at a higher level in human thyroid than in the heart or stomach29, but its role in the thyroid has not previously been reported. Furthermore, 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 described30'31.
[00039] T3 and T4 biosynthesis requires active Γ transport in the thyroid, where Γ 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 Γ into the thyrocyte; at the cell-colloid interface, Γ ion is oxidized and covalently incorporated into thyroglobulin for thyroid hormone production17. 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. Here we identify KCNQ1-KCNE2 as a TSH- stimulated thyrocyte K+ channel crucial for normal thyroid Γ accumulation and probably expressed predominantly at the basolateral membrane.
[00040] The marked effects of surrogacy in the current study add to the debate over whether maternal T4 is at high enough concentrations in milk to deliver therapeutic effects in hypothyroxinemic newborns32. Our findings suggest that, at least in mice, T4 is at a high enough concentration in milk to be potentially therapeutic for hypothyroxinemic pups. Maternal Kcne2 deficiency reduced the amount of T4 in milk and impaired milk ejection, with both defects likely contributing to the poor development of pups feeding from Kcne2~'~ dams. The mechanisms underlying the whole-animal and molecular effects of surrogacy seem to be complex, as one would expect. Kcne2~'~ pups are less efficient at accumulating thyroid Γ compared to Kcne2+I+ pups when both are fed by Kcne2+I+ dams, but have similar ability to accumulate thyroid Γ when fed by Kcne2~'~ dams. We speculate that a defect in thyroid Γ accumulation in Kcne2~'~ pups is partially balanced by other factors, including adaptation to their development in a low maternal T4 environment in the womb and their being initially fed with poorly ejected, low-T4 milk. Part of this adaptation may involve reduced Γ excretion by Kcne2~'~ pups, consistent with previous reports showing reduced Γ 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 models33. 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.
[00041] As is also the case for NIS16'34, KCNQl is expressed in both thyroid and mammary gland epithelium; in the mammary gland, KCNQl may assemble with KCNE3 to contribute to K+ homeostasis35. Although a role for KCNE2 in mammary epithelial function should not be ruled out, our PET data indicate that mammary gland Γ uptake is not impaired in Kcne2~'~ dams.
[00042] Human thyroid dysfunction negatively affects the brain, heart and gastrointestinal tract; fatalities may occur from thyroid storm in hyperthyroidism and myxedema coma in hypothyroidism36. In addition, thyroid dysfunction during pregnancy increases the risk of adverse maternal and fetal outcomes37 39. Subclinical human maternal hypothyroxinemia causes severe neurodevelopmental disorders40, may cause changes in blood lipid profile, myocardial function and neuropsychiatric function41 43, and is an independent risk factor in heart failure due to structural and electrical remodeling in the heart24. Notably, a single nucleotide polymorphism near KCNE2 was recently shown to associate with early -onset myocardial infarction44, suggesting the possibility of a genetic link to previously reported subclinical hypothyroidism-associated accelerated coronary artery disease and myocardial infarction45.
[00043] Subclinical hypothyroidism is also associated with a prolonged corrected
QT interval (QTc) on the electrocardiogram46, a hallmark of loss-of- function mutations in KCNE2 and KCNQl2,6, and with atrial fibrillation, an increasingly prevalent disease in the aging population47'48 that is also associated with some KCNQl and KCNE2 gene variants9'12. As many as 13% of individuals with idiopathic atrial fibrillation show biochemical evidence of hyperthyroidism49, and, in one study, 62% of 163 subjects reverted to sinus rhythm within 8-10 weeks after treatment for hyperthyroidism returned them to a euthyroid state50. The finding that KCNE2-KCNQ1 channels contribute to thyroid function raises the hypothesis that there is a thyroid component to some KCNE2- or KCNQl -associated cardiac arrhythmias. In previous studies of sudden cardiac or unexplained death, it was often assumed that ion channel gene mutations were not causative in those cases showing overt structural heart disease upon autopsy51.
Historically, 'electrical' heart diseases arising from ion channel defects have mostly been considered genetically distinct from 'structural' heart disease, although variants in the gene encoding the human SCN5A Na+ channel have been associated with dilated cardiomyopathy52. Our findings suggest consideration of the possibility of a shared genetic basis for structural heart disease and cardiac arrhythmias in individuals exhibiting both,given the possibility that mutations in KCNQl and KCNE2 could be
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.
[00044] Identification of KCNE2-KCNQ 1 as a thyrocyte channel crucial for Γ accumulation may also have therapeutic implications. Agonists and antagonists of KCNQl -KCNE2 channels have already been developed. Because the pharmacology of KCNQl -KCNE2 complexes is markedly different from that of homomeric KCNQl, KCNQl -KCNE1 or KCNQl -KCNE3 channels53, identification of the requirement for KCNQl -KCNE2 complexes for normal thyroid function may permit semispecific, reversible pharmacological targeting of the KCNQl -KCNE2 complex to treat thyroid disease.
[00045] Observing Whether a Genome Contains a KCNQl or KCNE2 Allele Having an Alteration
[00046] 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.
[00047] The genome of a patient generally contains two each of the KCNQl and
KCNE2 alleles. An allele, as used herein, is 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.
[00048] The term "genetic alteration," as used herein, 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. Accordingly, 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. Examples of 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.
[00049] Accordingly, the genetic alteration can occur at any nucleotide position(s) in the nucleotide sequence of KCNQl or KCNE2. For example, the genetic alteration can occur at the beginning, middle or end of the nucleotide sequence.
[00050] 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.
[00051] A nucleotide substitution refers to the replacement of a nucleotide with a different nucleotide. An example of a substitution is a single nucleotide polymorphism.
[00052] 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.
[00053] 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.
[00054] For example a gain of function mutation in either gene causes atrial fibrillation or short QT syndrome, while a loss of function causes Long QT syndrome.
[00055] Such mutations are known in the art, for example, see McCrossan et al
(2009); Webster G, Berul CI. (2008); Shimizu W. (2008); Schimpf R, et al (2008); and Peroz D, et al (2008) , each incorporated herein by reference.
[00056] A genetic alteration may occur within one copy or both copies of a
KCNQl or KCNE2 allele. 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.
Alternatively, 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.
[00057] The patient's KCNQl or KCNE2 genotype can be homozygous or heterozygous for any genetic alteration, such as those mentioned above. For example, a patient may be homozygous or heterozygous for any SNP.
[00058] The determination of an allele having a genetic alteration can be made by any method known to those skilled in the art. Suitable methods are provided in the "General Methods" section below.
[00059] In another embodiment, the determination of a genetic alteration comprises observing expression of a KCNQ 1 or KCNE2 protein containing an amino acid alteration.
[00060] The term "amino acid alteration" refers to any changes in the amino acid sequence of wild-type KCNQl or KCNE2 protein. Thus, KCNQl or KCNE2 proteins that contain an amino acid alteration will have a different amino acid sequence than wild- type KCNQl or KCNE2 protein. Examples of 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.
[00061] 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. One type of 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.
[00062] The observation of expression of a KCNQ1 or KCNE2 protein having an amino acid alteration can be made by any method known to those skilled in the art.
Suitable methods are provided in the "General Methods" section below.
[00063] Correlating the Presence of a Genetic Alteration with Susceptibility or Resistance
[00064] The term "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.
[00065] In a subject, the presence of a KCNQ1 or KCNE2 allele having a genetic alteration is correlated with susceptibility to or having a thyroid disease.
[00066] General Diagnostic Methods
[00067] To observe whether the genome of a patient contains at least one copy of the KCNQ1 or KCNE2 allele containing a genetic alteration, a sample containing the patient's DNA is obtained. Examples of such samples include blood, salvia, urine and epithelial cells.
[00068] 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.
[00069] DNA can be isolated from the sample by any method known to those in the art. For example, commercial kits, such as the QIAGEN System (QIAmp DNA Blood Midi Kit, Hilder, Germany) can be used to isolate DNA. [00070] 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.),
Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001). For example, 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.
[00071] 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.
[00072] Alternative methods include, for example, use of 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).
[00073] For example, methods for distinguishing a wild-type allele from an allele containing a single nucleotide change are described in PCT Application WO 87/07646. The methods disclosed in PCT Application WO 87/07646 are incorporated herein by reference.
[00074] Briefly, 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
endonuclease. An example of suitable stringent conditions includes a temperature of two or more degrees below the calculated Tm of a perfect duplex. The oligonucleotide probe hybridizes to the target DNA or RNA detectably better when the probe and the target are perfectly complementary.
[00075] A particularly convenient method for assaying a single point mutation by means of oligonucleotides is described in Segev, PCT Application WO 90/01069. The methods disclosed in PCT Application WO 90/01069 are hereby incorporated by reference.
[00076] Briefly, two 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.
[00077] 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. Either of the oligonucleotides or the nucleotide filling the gap may be labelled by methods known in the art.
[00078] 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.
[00079] The oligonucleotide product can be separated by size and the label is detected by methods known in the art.
[00080] 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).
[00081] For example, point mutations can be detected by means of single-strand conformation analysis of polymerase chain reaction products (PCR-SSCP). This method is described in Orita, M. et al, Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989), Suzuki, Y. et al, Oncogene 5:1037-1043 (1990), and Sarkar, F.H. et al, Diagn. Mol. Pathol. 4:266-273 (1995).
[00082] Some additional methods for distinguishing a wild-type allele and allele containing a genetic alteration are described by De Ley et al., J. Bacteriol. 101 :738-754 (1970); Wood et al, Proc. Natl. Acad. USA 82: 1585-1588 (1985); Myers et al, Nature 313:495-497 (1985); and Myers et al, Science 230: 1242-1246 (1985).]. See also U.S. Patent Application Publication No. 2005/0014170, which discloses assays for observing A KCNQ1 OR KCNE2 genotypes, the specification of which is hereby incorporated by reference.
[00083] To observe whether the patient expresses a KCNQ1 or KCNE2 protein containing an amino acid alteration, 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.
[00084] Protein can be isolated from the sample by any method known to those in the art. For example, commercial kits, such as the Mono Q ion exchange
chromatography (Amersham Biosciences, Piscataway, NJ) can be used to isolate the protein.
[00085] 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.
[00086] Briefly, 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.
[00087] 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
Campbell in "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); as well as the recombinant DNA method described by Huse et al. in Science 246, 1275-1281 (1989).
[00088] In order to produce monoclonal antibodies, 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). In order to be useful, 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).
[00089] 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.
[00090] Thyroid disease
[00091] Hyperthyroidism
[00092] Too much thyroid hormone from an overactive thyroid gland is called hyperthyroidism, because it speeds up the body's metabolism.
[00093] Because the thyroid gland is causing a hormone imbalance by producing too much hormone in hyperthyroidism, the body develops an increased metabolic state, with many body systems developing abnormal function.
[00094] 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
[00095] 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.
[00096] 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.
[00097] 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.
[00098] Other causes of hyperthyroidism include: Getting too much iodine;
Inflammation (irritation and swelling) of the thyroid due to viral infections or other causes; Non-cancerous growths of the thyroid gland or pituitary gland; Taking large amounts of thyroid hormone; Tumors of the testes or ovaries
[00099] Mutations that cause gain of function in KCNQ1 or KCNE2 can cause, or create susceptibility to, hyperthyroidism.
[000100] Drugs that block KCNQ 1 or KCNE2, or that block KCNQ 1 -KCNE2 channels, are useful for treating hyperthyroid diseases.
[000101] Hypothyroidism
[000102] Too little thyroid hormone from an underactive thyroid gland is called hypothyroidism, another hormone imbalance caused by thyroid problems. In
hypothyroidism, the body's metabolism is slowed. Several causes for this condition exist, most of which affect the thyroid gland directly, impairing its ability to make enough hormone. More rarely, there may be a pituitary gland tumor (located near the base of the brain), which blocks the pituitary from producing thyroid-stimulating hormone (TSH). As a consequence, the thyroid fails to produce a sufficient supply of hormones needed for good health.
[000103] Whether 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.
[000104] Symptoms
[000105] 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.
[000106] This thyroid disease's symptoms include the following: Fatigue; Mental depression; Sluggishness; Feeling cold; Weight gain; Dry skin and hair; Constipation; Menstrual irregularities [000107] 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
[000108] The most common cause of hypothyroidism is 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").
[000109] 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.
[000110] Mutations that cause loss of function in KCNQ1 or KCNE2 will cause, or create susceptibility to, hypothyroidism.
[000111] Compounds that promote the activity of KCNQ1 or KCNE2, or that open KCNQ1-KCNE2 channels, are useful for treating hyperthyroid diseases.
[000112] Compounds:
[000113] 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.
[000114] Subject:
[000115] As used herein, the term "subject" is intended to include human and non- human animals. 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 african, asian, Semitic, or of other or mixed racial background. Preferred subjects include human patients suffering from or at risk for thyroid disease.
Examples
[000116] Transgenic mice generation, care and use. We generated and genotyped the Kcne2~'~ mouse line as previously described15'19 and housed and used mice according to the US National Institutes of Health Guide for the Care and Use of
Laboratory Animals. Animal procedures were approved by the Animal Care and Use Committee at Weill Medical College of Cornell University or at Albert Einstein College of Medicine.
[000117] Human Tissue. Human thyroid tissue slides were obtained from a commercial source (ProSci) after informed consent was obtained.
[000118] Measurement of tetraiodothyronine, thyroid-stimulating hormone and iodide. We killed mice by carbon dioxide asphyxiation. We collected whole blood postmortem by cardiac puncture. After coagulation of the remaining whole blood at 23 °C for 30 min, we centrifuged the clotted blood at 800 g for 5-10 min at 4 °C. We collected milk by hand 2 h after injection of pup-separated, lactating dams with 20 μg per g body weight oxytocin (Sigma). We quantified plasma T4 and TSH by enzyme-linked immunoassay (AniLytics, Inc.). We quantified T4 in pregnant mouse plasma and in milk with the Siemens Total T4 coat-a-count radioimmunoassay (Endocrinology Laboratory of Cornell University Animal Health Diagnostic Center). We measured plasma Γ was measured by a previously described colorimetric assay54.
[000119] Thyroid hormone administration and surrogacy. For thyroid hormone administration, we injected pups intraperitoneally every other day with 130 ng T4 and 13 ng T3 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 μg per g body weight T3 and 0.4 μg per g body weight T4 for 2 weeks. We fed pregnant dams T4 ad libitum at 5 mg Γ1 in their drinking water, from the last 2 weeks of gestation to the weaning of their pups, with dosage and preparation as previously described55. For PET imaging of 124I transfer from dams to pups, we established surrogates 1-2 d before imaging and confirmed latching on before initiation of imaging. For milk ejection studies, we established surrogates only for the 1-h feeding period. For all other surrogacy experiments, we established surrogates at postnatal day 0.
[000120] Electrophysiology. We recorded K+ channel activity in cultured FRTL5 cells (American Type Culture Collection) by whole-cell patch-clamp recordings at 22- 25 °C using an inverted microscope 1X50 (Olympus), a Multiclamp 700A amplifier, a Digidata 1300 analog/digital converter and a computer with pCLAMP9.2 software (Axon Instruments). We bathed the cells in a physiological solution of 140 mM NaCl, 5.4 mM KC1, 1.8 mM CaCl, 0.5 mM MgCl2, 5 mM HEPES and 5 mM glucose (pH 7.4). We obtained XE991 -sensitive currents by digital subtraction of current after application of 10 μΜ XE991 (Tocris Bioscience) from the current from the same cell before XE991 application. Borosilicate glass pipettes (Sutter) were of 3-5 ΜΩ resistance when filled with intracellular solution containing 120 mM potassium aspartate, 20 mM KC1, 1 mM MgCl2, 5 mM HEPES, 0.5 mM EGTA and 1 mM CaCl2 (pH 7.2 with KOH). We stepped the cells from a holding potential of -40 mV to test potentials between -120 mV and 60 mV in 20-mV increments for a 2-s duration at 0.1 Hz. We analyzed data with pClamp9 software (Axon Instruments) and analyzed statistics where appropriate with Origin (Microcal) software. We assessed K+ channel activity in cardiac myocytes after isolation of ventricular myocytes using the Langendorff perfusion system and culturing of myocytes for less than 12 h as previously described19.
[000121] Positron emission tomography. We gave lactating dams anesthetized with 1.5% isoflurane-oxygen mixture 200-300 μ& 124I in 0.1 ml normal saline via tail vein injection. After injection of tracer and the initial 1-h dynamic imaging, we placed the dams back in their cages to permit the nursing of their pups over the next 3-4 d. We imaged the dams and pups at various times, as described in Figs. 5 and 6 and
corresponding text. We determined the amount of radioactivity ingested by each pup by measurement of the entire pup in the dose calibrator before each imaging session.
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. We performed list mode acquisition of data for the first hour to permit dynamic reframing for kinetic evaluation of 124I uptake. We obtained delayed images of 10-min duration on the day of injection and on subsequent days up to 96 h after 124I administration. After each acquisition, we sorted data into three- dimensional sinograms and reconstructed images with iterative reconstruction in a 128 x 128 x 64 pixel (0.82 x 0.82 x 1.2 mm) array. We corrected data for dead-time counting losses, random coincidences and the measured nonuniformity of detector response (that is, normalized) but not for attenuation or scatter.
[000122] We analyzed the data with ASIPRO (Siemens) dedicated software. We inspected all image studies visually in a rotating three-dimensional projection display to examine for interpretability and image artifact. We manually defined regions of interest around areas identified as thyroid, mammary gland, and stomach. Successive scrolling through two-dimensional slices (each 1.2 mm thick in the axial images) permitted both measurement of radioactivity within defined volumes and visualization of radioactivity profiles in reconstructed image planes. After correction for detector sensitivity, radioactivity was measured as the counts per ml within a region of interest multiplied by the three-dimensional volume of the region. Ratios of radioactivity in various organs could then be determined.
[000123] Milk ejection studies. As previously described27, we separated lactating dams from their pups for 8 h and then weighed the pups and distributed them among dams so that each would feed eight pups: four Kcne2+I+ pups and four Kcne2~'~ pups. We weighed the pups after 30 and 60 min feeding. The difference in mass (30- or 60-min mass minus 0-min mass) equaled the mass of milk ingested less the mass of any excreta. Where indicated, we injected oxytocin (20 μg per g body weight; Sigma) into dams 10 min before uniting dams with pups.
[000124] Statistical analyses. We analyzed numerical data with Excel (Microsoft) or Origin (Microcal) software using ANOVA with statistical significance set at P < 0.05. [000125] Electron Microscopy, Histology and Immunofluorescence For electron microscopy, thyroid tissue from Kcne2+I+ and Kcne2~'~ mice (2/gender/genotype) was washed, fixed, stained, and dehydrated and then infiltrated and embedded in Spurr's resin. Sections were cut, contrasted with lead citrate, and viewed on a JSM 100 CX-II electron microscope (JEOL USA Inc., Peabody, MA) operated at 80 kV. Images were recorded on Kodak 4489 Electron Image film and then digitized on an Epson Expression 1600 Pro scanner at 900 dpi. For histology, Kcne2+I+ and Kcne2~'~ mice were sacrificed using C02 asphyxiation. Thyroids were removed, fixed in 10% neutral buffered formalin, processed by routine methods, and frozen. Sections (5 μιη) were placed on super frost (positively charged) slides, stained with hematoxylin and eosin, and evaluated with an Olympus BX45 light microscope (New York/New Jersey Scientific, Inc., Middlebush, NJ).
Masson's Trichrome staining of cardiac and hepatic sections was performed to detect collagen deposition.
[000126] Hyperplastic human thyroid tissue slides from paraffin embedded tissue (ProSci) were used for immunofluorescence microscopy. Murine thyroid glands were frozen before sectioning. Immunofluorescence detection of NIS, KCNQ1 and KCNE2 was performed using a Discovery XT processor (Ventana Medical Systems). 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 μg/ml. Preceding the primary antibody incubation, 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). [000127] Western Blotting Post mortem, thyroids from 20 adult Kcne2 and Kcne2~'~ mice were removed and snap-frozen in liquid N2; pooled tissue samples from both genotypes were dounze homogenized in ice-cold homogenization buffer (280 mM mannitol, 10 mM HEPES, 10 mM KC1, 1 mM MgC12, pH 7.0 with Tris, plus protease inhibitor cocktail (Roche)) and spun at 2,500 g for 5 minutes to remove cell debris and nuclear material. This spin and the following spins were carried out at 4 °C. The supernatant was further homogenized and spun at 11,000 x g for 20 minutes. The pellet was discarded and the supernatant was spun at 150,000 x g for 90 minutes. The pellet was resuspended in 200 μΐ of homogenization buffer and the protein concentration was measured according to the Bradford method then normalized to total protein
concentration before loading (40 μg total protein/lane) into pre-cast tris-glycine 4-20% or or 13% gels and separation by electrophoresis. Proteins were then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA), and blocked with 0.05% Tween-20 in PBS or TBS (Buffer A) with 5% milk for 4°C on a rocker overnight. Primary antibody incubations (4 hr, RT in Buffer A with 1% milk) were: 1 :2000 anti-rat-KCNE2 (Sigma- Aldrich # M3318 or in-house15); 1 : 1000 anti-KCNQl (Santa Cruz Biotech # #SC-10646); 1 g/ml rabbit polyclonal anti-rat-NIS (in-house). Membranes were washed 4 times, 20 min each with antibody incubation buffer then incubated with the appropriate secondary antibodies (BioRad) diluted 1 : 10,000 in Buffer A for 2 hr at RT, then washed 4 x 20 min each with Buffer A and once for 5 min with PBS. 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
downstream effector of TSH) before cell harvesting and lysis.
[000128] Cell culture FRTL5 cells were grown on uncoated glass coverslips in Coon's modified Ham's F12 medium supplemented with 5% newborn calf serum
(Sigma) and six different hormones (insulin 10 μg/ml, transferrin 5μg ml"1,
hydrocortisone 10 nM, tripeptide gly-L-his-L-lys 10 ng ml"1, TSH 0.3 mU ml"1, and somatostatin 10 ng ml"1) in a watersaturated atmosphere of 5% C02 and 95% air at 37 °C. 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.
[000129] 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. Left ventricular (LV) fractional shortening (FS) was calculated from the M-mode recordings using the equation FS (%) = (LVEDD-LVESD)/LVEDD x 100.
[000130] The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application) are hereby expressly incorporated by reference. These examples are directed to testing the efficacy of sildenafil using a shoulder-elbow motion device but could be readily adapted to testing other drugs and other therapies using other attachments.
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Claims

1. A method to treat hyperthyroid disease by administering to a subject in need thereof a compound known to block the activity of KCNQl or KCNE2 or that blocks the KCNQl -KCNE2 channel.
2. A method to treat hypothyroid disease by administering a compound known to promote the activity of KCNQl or KCNE2 or that opens the KCNQl -KCNE2 channel.
3. A method to diagnose thyroid disease or determine a susceptibility to developing thyroid disease comprising:
i) taking a patient sample;
ii) measuring said patient sample to identify a mutation in KCNQl or KCNE2; and iii) determining whether said mutation is a loss of function mutation or a gain of function mutation.
4. The method of claim 3 further comprising correlating a gain of function mutation with a need to promote the activity of KCNQl or KCNE2 or open the KCNQl -KCNE2 channel; and a loss of function mutation with a need to block the activity of KCNQl or KCNE2 or block the KCNQl -KCNE2 channel.
5. The method of claim 4 further comprising reporting said correlation to a physician.
6. The method of claim 4 further comprising administering to a patient in need thereof a compound with the recommended activity.
PCT/US2010/049488 2009-09-18 2010-09-20 Kcnq1 and kcne2 in thyroid disease Ceased WO2011035239A1 (en)

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