WO1998029431A1 - Procede pour diagnostiquer et traiter des troubles pathologiques lies a une insuffisance du transport d'ions - Google Patents

Procede pour diagnostiquer et traiter des troubles pathologiques lies a une insuffisance du transport d'ions Download PDF

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WO1998029431A1
WO1998029431A1 PCT/US1997/023553 US9723553W WO9829431A1 WO 1998029431 A1 WO1998029431 A1 WO 1998029431A1 US 9723553 W US9723553 W US 9723553W WO 9829431 A1 WO9829431 A1 WO 9829431A1
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protein
tsc
romk
nkcc2
human
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PCT/US1997/023553
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Richard P. Lifton
David B. Simon
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Yale University
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Priority to AU60135/98A priority Critical patent/AU746220B2/en
Priority to EP97954793A priority patent/EP1030858A1/fr
Priority to JP53012398A priority patent/JP2001508291A/ja
Priority to CA002274955A priority patent/CA2274955A1/fr
Publication of WO1998029431A1 publication Critical patent/WO1998029431A1/fr

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders

Definitions

  • the invention relates to the fields of detecting and treating homozygous and heterozygous genetic deficiencies in ion transport, particularly alterations in nucleic acid molecules and proteins that give rise to various forms of Bartter's Syndrome and Gitelman's Syndrome. More specifically, the invention provides compositions and methods for determining whether an individual is affected by or carriers a mutation in one or more genes involved in ion transport.
  • kidney plays a dominant role in determining the long-term set points of fluid and electrolyte balance, maintaining homeostasis despite wide variation in environmental exposure. Derangements in these components of kidney function are likely to underlie a number of clinical disorders ranging from altered blood pressure due to changes in intravascular volume to abnormalities in electrolyte homeostasis. Examination of mendelian disorders of fluid and electrolyte homeostasis provides the opportunity to dissect the fundamental mechanisms governing this process.
  • Bartter's Syndrome is an autosomal recessive disorder featuring hypokalaemic metabolic alkalosis with salt wasting (Bartter, F.C., et al, Am. J. Med. 33:811-828 (1962)).
  • Affected patients have been shown to have a diverse array of additional metabolic abnormalities, including elevated plasma renin activity (Bartter, F.C., et al, Am. J. Med.
  • Bartter's Syndrome has been proposed to be a heterogeneous entity with at least two subsets, Gitelman's Syndrome (Gitelman, H.J., Graham, J.B., and Welt, L.G. A new familial disorder characterized by hypokalaemia and hypomagnesemia. Trans. Assoc. Am. Phys. 79:221-235 (1966)) and "true Bartter's Syndrome" (Bettinelli, A., et al, J. Pediatr. 120:38-43 (1992)).
  • Gitelman's Syndrome refers to the predominant subset of patients with hypokalaemic alkalosis in conjunction with hypocalciuria and hypomagnesemia, while true Bartter's Syndrome refers to patients with normal or hypercalciuria and typically normal magnesium levels. True Bartter's patients are said to present clinically at early ages (less than 5 years) with signs of vascular volume depletion, while Gitelman's Syndrome patients typically present at older ages without overt hypovolemia (Bettinelli, A., et al, J. Pediatr. 120:38-43 (1992)).
  • TSC thiazide-sensitive cotransporter
  • TSC thiazide-sensitive cotransporter
  • This cotransporter is the target of thiazide diuretics, one of the major classes of agents used in the treatment of high blood pressure.
  • cDNAs encoding the TSC have recently been cloned from flounder bladder and rat kidney (Gamba, G., et al, Proc. Natl.
  • the encoded protein from rat comprises 1002 amino acids, and contains twelve putative transmembrane domains, with long intracellular amino and carboxy termini. Similarities in some features of patients with Gitelman's Syndrome and patients receiving thiazide diuretics raise the possibility that mutation in TSC causing loss of function could result in Gitelman's Syndrome. This consideration motivates examination of TSC as a candidate gene for Gitelman's Syndrome.
  • Gitelman's Syndrome is a genetically homogeneous autosomal recessive trait caused by loss of function mutations in the thiazide-sensitive Na-Cl cotransporter protein (TSC) located in the renal distal convoluted tubule.
  • TSC thiazide-sensitive Na-Cl cotransporter protein
  • NKCC2 also known as SLC12A1
  • SLC12A1 The absorptive variant of the bu etanide- sensitive Na-K-2C1 cotransporter (NKCC2 , also known as SLC12A1) is the primary mediator of sodium and chloride reabsorption in this nephron segment (Greger, R., Physiol. Rev. 65:760-797 (1985)), and loss of function of this cotransporter could produce many of the features seen in affected patients.
  • loop diuretics can produce electrolyte disturbances very similar to those seen in patients with Bartter's Syndrome (Greger, R., et al, Klin. Woschenschr. 61 :1019-1027 (1991)).
  • cDNA's encoding NKCC2 have recently been cloned from rat (Gamba » £j. et al, J. Biol. Chem. 26:17713-17722 (1994)), rabbit (Payne, J.A., et al, Proc. Natl. Acad. Sci. (U.S.A.) 91 :4544-4548 (1994)) and mouse (Igarashi, P. et al, Am. J.
  • NKCCl also known as SLC12A2
  • shark Xu, J.-C. et al, Proc. Natl. Acad. Sci. (U.S.A.) 91:2201-2205 (1994)
  • human Payne, J.A. et al, J. Biol. Chem. 270:17977-17985 (1995)
  • All members of this family have 12 putative transmembrane spanning domains and also show structural and sequence similarity to TSC.
  • Example 2 demonstrates that this variant of inherited hypokalaemic alkalosis is caused by mutations in the gene encoding NKCC2.
  • NKCCl locus symbol SLC12A1
  • TAL Henle's loop
  • Bartter's Syndrome featuring salt wasting and hypokalaemic alkalosis associated with marked hypercalciuria and frequently nephrocalcinosis
  • Bartter's patients typically are born prematurely with polyhydramnios and show marked dehydration in the neonatal period
  • Gitelman's patients typically present at older ages with neuromuscular signs and symptoms (Wang, W., et al, Ann. Rev. Physiol. 54:81-96 (1992)).
  • K + channel An inwardly rectifying K + channel (IRK) bearing many features of this regulatory channel (low single channel conductance, activation by low levels of ATP and protein kinase A (PKA), and insensitivity to voltage and calcium) has been cloned (Ho, K., et al, Nature 362:31-38 (1993)).
  • This channel, ROMK locus symbol KCNJ1
  • ROMK locus symbol KCNJ1
  • the channel contains PKA phosphorylation sites that are required for normal channel activity.
  • ROMK isoforms encoded by the same chromosome 11 locus are generated by alternative splicing (Yano, H., et al, Mol. Pharmacology 45:854-860 (1994); Shuck, M.E. et al, J. Biol. Chem. 269:24261- 24270 (1994)); these isoforms have been shown to be expressed in the kidney, specifically on the apical membrane of cells of the TAL as well as more distal nephron segments (Lee, W.S., et al, Am. J. Physiol. (Renal Fluid Electrol. Physiol.) 268 :F1124-31 (1995); Boim, M.A. et al, Am. J.
  • the present invention provides compositions and methods that can be used to differentiate and diagnose several types of ion transport deficiencies, particularly Bartter's Syndrome and Gitelman's Syndrome.
  • the present invention further provides methods and compositions that can be used to identify heterozygote carriers for these disorders. Carriers, though not displaying severe clinical symptoms, nonetheless display mild to moderate pathologies.
  • the present invention is based, in part, on the identification of the roles of the human thiazide-sensitive Na-Cl cotransporter, TSC; the human ATP-sensitive K + channel, ROMK; and the human Na-K-2C1 cotransporter, NKCC2 in pathological condition associated with abnormal ion transport, particularly Bartter's Syndrome, Gitelman's Syndrome, hypokalaemic alkalosis, hypokalaemic alkalosis with hypercalciuria, kidney stones, high blood pressure, osteoporosis and sensitivity to diuretic-induced hyperkalaemia.
  • the present invention specifically provides the amino acid sequences of several human wild-type and altered variants of the TSC, NKCC2 and ROMK proteins as well as the nucleotide sequence that encodes these variants. These proteins and nucleic acid molecules can be used in diagnosing ion transport disorders and in developing methods and agents for treating these pathologies.
  • Gitelman's Syndrome kindreds used for linkage studies The familial relationships of Gitelman's Syndrome kindreds used for linkage studies are shown. Individuals with Gitelman's Syndrome are indicated by filled symbols; individuals who do not have Gitelman's Syndrome are indicated by unfilled symbols; deceased individuals are indicated by a diagonal line through the symbol. Individuals not sampled for genetic studies are indicated by a dot within the symbol. Each kindred is given a unique kindred number, and each individual within the kindred is numbered above and to the left of the symbol. Below each symbol, genotypes at loci on chromosome 16 are shown in their map order (see Figure 3 for map of loci).
  • TSC SSCP refers to variants identified in TSC; different variants are given different allele numbers and the nature of each variant is indicated in Table 1.
  • SSCP allele 1 represents the wild-type SSCP variant. Inferred haplotypes cosegregating with Gitelman's Syndrome are enclosed by boxes, with maternal and paternal haplotypes distinguished by shaded or unfilled boxes, respectively.
  • Cosmid contig spanning the TSC locus Thin horizontal bars represent cloned human genomic DNA of cosmid clones; vertical bars indicate sites cleaved by restriction endonuclease EcoRI. Independent clones are drawn with their overlaps indicated, and the 5' to 3' orientation of transcription is shown.
  • the multipoint lod score for linkage of Gitelman's Syndrome across this interval is shown, revealing a lod score of 9.5 at a recombination fraction of zero with D16S408, and showing odds of greater than 1000:1 favoring location of the trait locus in the 11 centimorgan interval defined by flanking loci D16S419 and D16S494.
  • the lod-1 support intervals for the location of the TSC locus defined in CEPH kindreds and for the location of the Gitelman's locus in disease kindreds are shown, revealing that they overlap.
  • molecular variants in TSC show linkage to Gitelman's Syndrome at a recombination fraction of zero (see Figure 1).
  • FIG. 4 Novel variants in patients with Gitelman's Syndrome. Variants in patients with Gitelman's Syndrome were identified by SSCP and subjected to DNA sequence analysis as described in Methods. Representative examples are shown. Autoradiograms of variants detected on non-denaturing gels (panels A,B,C,E,F) or denaturing gels (panel D) are shown at the left of each panel; patients are identified as in Figure 1, and subjects with Gitelman's Syndrome are indicated by asterisks; arrows indicate variants specific for Gitelman's Syndrome kindreds. At the right of each panel the DNA sequences of the corresponding variant (top) and wild-type (bottom) are shown.
  • Variant bases are indicated by an asterisk; in panel D, the 3 bases in the wild-type sequence that are deleted in the variant are indicated by a bracket. All sequences are shown in the antisense orientation with respect to the gene. With the exception of panels D and E, 9 bases, corresponding to the mutated codon and the two flanking codons, are shown.
  • A Variant alters R209 to W in GIT 102; B, Variant alters P349 to L in GIT107; £ Variant alters C421 to R in GIT102; H Three base deletion changes sense sequence CCTTCA encoding PS561 to CCA, deleting codon 561 in GIT108; R Variant in sense orientation changes consensus 3' splice site CAG to CAT in intron 15 in GIT102. Variant alters R955 to Q in GIT11 1.
  • Figure 5. Schematic diagram of the TSC and mutations in Gitelman's Syndrome patients.
  • the TSC protein is represented as a 12 transmembrane domain protein with intracytoplasmic amino and carboxy termini (Gamba, G., et al, Proc. Natl.
  • FIG. 6 Bartter's Syndrome kindreds. Family relationships are shown. Affected subjects, unaffected subjects, living unsampled subjects and deceased subjects are indicated by filled symbols, unfilled symbols, dotted symbols and diagonal lines, respectively. Index cases are indicated by an arrow. Genotypes of loci tightly linked to NKCCl are indicated and are arranged in their chromosomal order (see Figure 7c); loci are identified to the left of kindred BAR152. Novel SSCP variants detected in NKCCl in each kindred are numbered, with the wild-type SSCP variant denoted by +. Affected offspring of consanguineous union are seen to be homozygous for all loci linked to NKCCl, and are homozygous for novel SSCP variants.
  • Figure 7 Characterization of the human genomic NKCCl locus. A. Intron-exon organization. Gray boxes indicate the 26 exons encoding the
  • NKCC2 protein The first codon in each exon is indicated; exons that begin with the second or third base of a codon are indicated by the subscript 2 or 3, respectively.
  • NKCC2 protein Single letter code. The sequence of the corresponding rat and shark sequence is shown beneath the human sequence. Amino acids that are identical to human residues are indicated by dots while residues that are different in these species are indicated. Transmembrane domains proposed from hydropathy plots are shaded and numbered Ml to Ml 2.
  • Figure 8 Novel variants in NKCCl in Bartter's Syndrome patients. Variants were identified by SSCP and subjected to DNA sequence analysis. Representative examples of autoradiograms are shown at the top of each panel, and the corresponding DNA sequence of the sense strand of wild-type (left) and mutant alleles (right) are shown at the bottom of each panel. Patients are numbered as in Figure 6, and subjects with Bartter's Syndrome are indicated by asterisks. The symbol nl represents unrelated normal subjects. Arrows indicate variants specific for Bartter's Syndrome kindreds. In panels a and b, brackets above the sequence figures indicate the positions of single base insertions or deletions. In panels d and e, variant bases are indicated by an asterisk above the sequence figures.
  • FIG. 9 Principal pathways and disorders altering renal sodium reabsorption.
  • a diagram of a nephron is shown. Plasma is filtered at the crescent-shaped structure representing the glomerulus, and sodium is reabsorbed as filtrate passes along the nephron.
  • the physiologic mediators of sodium reabsorption are indicated, and the fraction of filtered sodium that is normally reabsorbed by each pathway is indicated.
  • Disorders resulting from mutations in specific mediators of sodium reabsorption are indicated.
  • the principle mediators of sodium reabsorption are: Na + -H + exchange in the proximal tubule; Na-K-2C1 cotransport in the thick ascending limb of Henle; Na-
  • 58814 Cl cotransport in the distal convoluted tubule; electrogenic sodium reabsorption via the epithelial sodium channel, composed of at least 3 different subunits, in the distal nephron. This last pathway is indirectly coupled to secretion of K + and H + .
  • FIG. 10 Bartter's Syndrome kindreds. Family relationships are shown. Affected subjects, unaffected subjects, living unsampled subjects and deceased subjects are indicated by filled symbols, unfilled symbols, dotted symbols and diagonal lines, respectively. Index cases are indicated by an arrow. Genotypes of loci tightly linked to NKCC2 are indicated and are arranged in their chromosomal order; these 5 loci are linked within a 3 cM interval; GT7-3 is present on the same PAC clones as NKCC13. Below these genotypes, novel SSCP variants detected in ROMK in each kindred are numbered, and correspond to numbered variants in Figure 11; the wild-type SSCP variant is denoted by +.
  • NKCCl Linkage to NKCCl is seen to be excluded in the consanguineous kindreds BAR159 and BAR161.
  • novel ROMK variants are identified that cosegregate with the disease.
  • Figure 11 Novel variants in NKCCl in Bartter's Syndrome patients. Variants were identified by SSCP and subjected to DNA sequence analysis. Representative examples of autoradiograms are shown at the top of each panel, and the corresponding DNA sequence of the sense strand of wild-type (left) and mutant alleles (right) are shown at the bottom of each panel. Patients are numbered as in Figure 10, and subjects with Bartter's Syndrome are indicated by asterisks. The symbol nl represents unrelated normal subjects.
  • FIG. 10 Arrows indicate variants specific for Bartter's Syndrome kindreds, and are numbered as in Figure 10.
  • brackets above the sequence figures indicate the positions of base pair insertions or deletions.
  • variant bases are indicated by an asterisk above the sequence figures.
  • A A single base substitution changes codon TAC (Y60) to TAG (Stop ⁇ O) in BAR159; this mutation is homozygous in affected, but not unaffected kindred members.
  • a single base insertion produces a frameshift in codons 13-14 in BAR161 ; this mutation is homozygous in the affected member of this kindred.
  • ROMK2 A schematic diagram of ROMK2 is shown and depicted as spanning the plasma membrane twice with an H5 domain containing the channel pore (Ho, K., et al, Nature 362:31-38 (1993)). The locations and consequences of mutations identified in Bartter's patients are identified.
  • the present invention is based, in part, on the identification of the roles of the: human thiazide-sensitive Na-Cl cotransporter, TSC; the human ATP-sensitive K + channel, ROMK; and the human Na-K-2C1 cotransporter, NKCC2 in pathological conditions associated with abnormal ion transport, particularly Bartter's Syndrome, Gitelman's Syndrome, hypokalaemic alkalosis, hypokalaemic alkalosis with hypercalciuria, kidney stones, high blood pressure, osteoporosis and sensitivity to diuretic induced hyperkalaemia.
  • abnormal ion transport particularly Bartter's Syndrome, Gitelman's Syndrome, hypokalaemic alkalosis, hypokalaemic alkalosis with hypercalciuria, kidney stones, high blood pressure, osteoporosis and sensitivity to diuretic induced hyperkalaemia.
  • the present invention specifically provides the amino acid sequences of several human wild-type and altered variants of the TSC, NKCC2 and ROMK proteins as well as the nucleotide sequence that encodes these variants. These proteins and nucleic acid molecules can be used in diagnosing ion transport disorders and in developing methods and agents for treating these pathologies. II. Specific Embodiments
  • the art had identified: the amino acid sequence of one allelic variant of a presumably wild-type rat and a wild-type flounder thiazide- sensitive Na-Cl cotransporter protein (TSC); the amino acid sequence of several allelic variants of a presumably wild-type human ATP-sensitive K + channel protein (ROMK); and the amino acid sequence of one allelic variant of a presumably wild- type rat, a wild-type rabbit and a wild-type mouse Na-K-2C1 cotransporter protein (NKCC2).
  • TSC the amino acid sequence of one allelic variant of a presumably wild-type rat and a wild-type flounder thiazide- sensitive Na-Cl cotransporter protein
  • ROMK human ATP-sensitive K + channel protein
  • NKCC2 wild-type mouse Na-K-2C1 cotransporter protein
  • the present invention provides, in part, the amino acid sequences of several allelic variants of wild-type human TSC protein, wild-type human NKCC2 protein, altered variants of the human TSC protein that give rise to ion transport deficiencies, and altered variants of the human NKCC2 protein that give rise to ion transport deficiencies, altered variants of the human ROMK protein that give rise to ion transport deficiencies, as well as the nucleotide sequence of the encoding nucleic acid molecules.
  • the present invention provides the ability to produce previously unknown wild-type and altered variants of the human TSC, NKCC2 and ROMK proteins using the cloned nucleic acid molecules herein described.
  • a wild-type human TSC protein refers to a protein that has the amino acid sequence of a wild-type allelic variant of human TSC.
  • DNA sequencing was performed on DNA isolated from 50 unrelated healthy individuals to identify wild-type TSC encoding DNA molecules.
  • Figure 2 and Table 3 provide the amino acid sequences of several wild-type allelic variants of the human TSC protein.
  • the wild-type TSC proteins of the present invention include those specifically identified and characterized herein as well as allelic variants that can be isolated and characterized without undue experimentation following the methods outlined below. For the sake of convenience, all of the wild-type human TSC proteins of the present invention will be collectively referred to as the wild-type TSC proteins or the wild-type human TSC proteins of the present invention.
  • wild-type human TSC proteins includes all naturally occurring allelic variants of the human TSC protein that posses normal TSC activity.
  • wild-type allelic variants of the TSC protein may/will have a slightly different amino acid sequence than that specifically provided in Seq. ID Nos for the herein- described wild-type TSC proteins.
  • Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will posses the ability to transport Na, Cl, Ca, and Mg at levels equivalent to the wild-type TSC proteins herein described.
  • allelic variants of the wild-type TSC protein will contain conservative amino acid substitutions from the wild-type TSC sequences herein described or will contain a substitution of an amino acid from a corresponding position in a TSC homologue (a TSC protein isolated from an organism other than human such as the rat or flounder homologues).
  • Figure 2 and Table 3 identify conserved amino acid residues.
  • a mutated or altered human TSC protein refers to a protein that has the amino acid sequence of a mutated or altered allelic variant of human TSC.
  • Figure 4, Table 1 and Table 4 provide the amino acid sequences of several mutated or altered allelic variants of the human TSC protein.
  • the mutated or altered TSC proteins of the present invention include those specifically identified and characterized herein as well as allelic variants that can be isolated and characterized without undue experimentation following the methods outlined below. For the sake of convenience, all of the mutated or altered human TSC proteins of the present invention will be collectively referred to as the mutated or altered TSC proteins or the mutated or altered human TSC proteins of the present invention.
  • mutated or altered human TSC proteins includes all naturally occurring allelic variants of the human TSC protein that do not posses normal TSC activity. In general, mutated or altered allelic variants of the TSC protein may/will have a slightly to a radically different amino acid sequence than that specifically provided in Seq. ID Nos for the herein-described wild-type TSC proteins.
  • allelic variants of the mutated or altered TSC protein will lack or have a reduced ability to transport one or more of the ions that are transported by wild-type TSC.
  • allelic variants of the mutated or altered TSC protein contain: non-conservative amino acid substitutions from the wild-type sequences herein described, a substitution of an amino acid other than the amino acid found in a corresponding position in a TSC homologue (a TSC protein isolated from an organism other than human), a frame shift mutation, an insertion of a stop codon, or a deletion or insertion of one or more amino acids into the TSC sequence.
  • a wild-type human NKCC2 protein refers to a protein that has the amino acid sequence of a wild-type allelic variant of human NKCC2.
  • DNA sequencing was performed on DNA isolated from 50 unrelated, healthy individ ⁇ als to identify wild-type NKCC2 encoding DNA molecules.
  • Figure 6 provides the amino acid sequences of the only wild-type allelic variant of the human NKCC2 protein thus far identified. Variations were seen in intron regions but no variation has been observed in the exon regions.
  • the wild-type NKCC2 proteins of the present invention include the one specifically identified and characterized herein as well as allelic variants that can be isolated and characterized without undue experimentation following the methods outlined below.
  • wild-type human NKCC2 proteins of the present invention will be collectively referred to as the wild-type NKCC2 proteins or the wild-type human NKCC2 proteins of the present invention.
  • wild-type human NKCC2 proteins includes all naturally occurring allelic variants of the human NKCC2 protein that posses normal NKCC2 activity.
  • wild-type allelic variants of the NKCC2 protein may/will have a slightly different amino acid sequence than that specifically provided in Seq. ID Nos for the herein-disclosed wild-type NKCC2 proteins.
  • allelic variants though possessing a slightly different amino acid sequence than those recited above, will posses the ability to transport Na, Cl, K, and Ca at levels equivalent to the wild-type NKCCS proteins herein described.
  • allelic variants of the wild-type NKCC2 protein will contain conservative amino acid substitutions from the wild-type sequences herein described or will contain a substitution of an amino acid from a corresponding position in a NKCC2 homologue (a NKCC2 protein isolated from an organism other than human).
  • a mutated or altered human NKCC2 protein refers to a protein that has the amino acid sequence of a mutated or altered allelic variant of human NKCC2.
  • Figure 8 and Table 7 provide the amino acid sequences of several mutated or altered allelic variants of the human NKCC2 protein.
  • the mutated or altered NKCC2 proteins of the present invention include those specifically identified and characterized herein as well as allelic variants that can be isolated and characterized without undue experimentation following the methods outlined below. For the sake of convenience, all of the mutated or altered human NKCC2 proteins of the present invention will be collectively referred to as the mutated or altered NKCC2 proteins or the mutated or altered human NKCC2 proteins of the present invention.
  • mutated or altered human NKCC2 proteins includes all naturally occurring allelic variants of the human NKCC2 protein that do not posses normal NKCC2 activity.
  • mutated or altered allelic variants of the NKCC2 protein may/will have a slightly to a radically different amino acid sequence than that specifically provided in Seq. ID Nos for the herein-described wild-type NKCC2 proteins.
  • Mutated or altered allelic variants will be not be able to transport one or more of the ions that are transported by wild-type NKCC2 or will transport ions at a rate that is substantially lower than the wild-type proteins.
  • allelic variants of the mutated or altered NKCC2 protein contain: non-conservative amino acid substitutions from a wild-type sequences herein described, a substitution of an amino acid other than the amino acid found in a corresponding position in a NKCC2 homologue (a NKCC2 protein isolated from an organism other than human), a frame shift mutation, an insertion of a stop codon, or a deletion or insertion of one or more amino acids into the NKCC2 sequence.
  • a wild-type human ROMK protein refers to a protein that has the amino acid sequence of a wild-type allelic variant of human ROMK.
  • DNA from 50 unrelated, healthy individuals was sequenced to identify wild-type ROMK encoding DNA molecules.
  • the amino acid sequences of the only wild-type allelic variant of the human ROMK protein identified are disclosed in .
  • the wild- type ROMK proteins of the present invention include that specifically identified and characterized in the art as well as allelic variants that can be isolated and characterized without undue experimentation following the methods outlined below.
  • allelic variants that can be isolated and characterized without undue experimentation following the methods outlined below.
  • all of the wild-type human ROMK proteins of the present invention will be collectively referred to as the wild-type ROMK proteins or the wild-type human ROMK proteins of the present invention.
  • wild-type human ROMK proteins includes all naturally occurring allelic variants of the human ROMK protein that posses normal ROMK activity.
  • wild-type allelic variants of the ROMK protein will have a slightly different amino acid sequence than that specifically provided in Seq. ID Nos .
  • Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will posses the ability to be an ATP sensitive K transporter.
  • allelic variants of the wild-type ROMK protein will contain conservative amino acid substitutions from the wild-type sequences herein described or will contain a substitution of an amino acid from a corresponding position in a ROMK homologue (a ROMK protein isolated from an organism other than human).
  • a mutated or altered human ROMK protein refers to a protein that has the amino acid sequence of a mutated or altered allelic variant of human ROMK.
  • Figures 11 and 12 and Table 10 provide the amino acid sequences of several mutated or altered allelic variants of the human ROMK protein.
  • the mutated or altered ROMK proteins of the present invention include those specifically identified and characterized herein as well as allelic variants that can be isolated and characterized without undue experimentation following the methods outlined below. For the sake of convenience, all of the mutated or altered human ROMK proteins of the present invention will be collectively referred to as the mutated or altered ROMK proteins or the mutated or altered human ROMK proteins of the present invention.
  • mutated or altered human ROMK proteins includes all naturally occurring allelic variants of the human ROMK protein that do not posses normal ROMK activity. In general, mutated or altered allelic variants of the ROMK protein may/will have a slightly to a radically different amino acid sequence than that specifically provided in Seq. ID Nos for the herein-described wild-type ROMK proteins. Mutated or altered allelic variants will be not be able to transport one or more of the ions that are transported by wild-type ROMK.
  • allelic variants of the mutated or altered ROMK protein will contain: non-conservative amino acid substitutions from the wild-type sequences herein described, a substitution of an amino acid other than the amino acid found in a corresponding position in a ROMK homologue (a ROMK protein isolated from an organism other than human), a frame shift mutation, an insertion of a stop codon, or a deletion or insertion of one or more amino acids into the ROMK sequence.
  • TSC, NKCC2 and ROMK proteins of the present invention are preferably in isolated from.
  • a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the TSC, NKCC2 or ROMK protein from cellular constituents that are normally associated with the protein.
  • a skilled artisan can readily employ standard purification methods to obtain an isolated TSC, NKCC2 or ROMK protein. The nature and degree of isolation will depend on the intended use.
  • TSC, NKCC2 and ROMK encoding nucleic acid molecules makes it possible to generate defined fragments of the TSC, NKCC2 and ROMK proteins of the present invention.
  • fragments of the TSC, NKCC2 and ROMK proteins of the present invention are particularly useful in generating domain specific antibodies, in identifying agents that bind to a TSC, NKCC2 or ROMK protein and in identifying TSC, NKCC2 or ROMK intra- or extracellular binding partners.
  • Fragments of the TSC, NKCC2 and ROMK proteins can be generated using standard peptide synthesis technology and the amino acid sequences disclosed herein. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode fragments of the TSC, NKCC2 and ROMK proteins. Figures 2, 5, 7 and 12 and Tables 1, 3, 4, 7 and 10 identify amino acid residues that are altered from wild- type residues in the altered variants of the TSC, NKCC2 and ROMK1 proteins herein described. Fragments containing these residues/alterations are particularly useful in generating altered variant specific anti-TSC, NKCC2 or ROMK antibodies.
  • members of the TSC, NKCC2 and ROMK family of proteins can be used for, but are not limited to: 1) a target to identify agents that block or stimulate TSC, NKCC2 or ROMK activity, 2) a target or bait to identify and isolate binding partners that bind a TSC, NKCC2 or ROMK protein, 3) identifying agents that block or stimulate the activity of a TSC, NKCC2 or ROMK protein and 4) an assay target to identify TSC, NKCC2 or ROMK mediated activity or disease.
  • the present invention further provides antibodies that selectively bind one or more of the TSC, NKCC2 or ROMK proteins of the present invention.
  • the most preferred antibodies will bind to an altered variant of a TSC, NKCC2 or ROMK
  • Anti-TSC, NKCC2 or ROMK antibodies that are particularly contemplated include monoclonal and polyclonal antibodies as well as fragments containing the antigen binding domain and/or one or more complement determining regions.
  • Antibodies are generally prepared by immunizing a suitable mammalian host using a TSC, NKCC2 or ROMK protein, or fragment, in isolated or immunoconjugated variant (Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).
  • Figures 2, 5, 7 and 12 and Tables 1, 3, 4, 7 ad 10 identify several regions of the TSC, NKCC2 and ROMK proteins that have been shown to be mutated in various altered variants of the TSC, NKCC2 and ROMK proteins described herein. Fragments containing these residues are particularly suited in generating wild-type or mutated-variant specific anti-TSC, NKCC2 or ROMK antibodies.
  • Methods for preparing a protein for use as an immunogen and for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be effective.
  • Administration of the TSC, NKCC2 or ROMK immunogen is conducted generally by injection over a suitable time period and with use of a suitable adjuvant, as is generally understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
  • Immortalized cell lines which secrete a desired monoclonal antibody may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the TSC, NKCC2 or ROMK protein or peptide fragment.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of the transporter can also be produced in the context of chimeric or CDR grafted antibodies of multiple species origin.
  • anti-TSC, NKCC2 or ROMK antibodies are useful as modulators of TSC, NKCC2 or ROMK activity, are useful in immunoassays for detecting TSC, NKCC2 or ROMK expression/activity and for purifying wild-type and altered variants of the TSC, NKCC2 and ROMK proteins.
  • the present invention is based, in part, on isolating nucleic acid molecules from humans that encode wild-type or altered variants of the TSC, NKCC2 and ROMK proteins. Accordingly, the present invention further provides nucleic acid molecules that encode the herein disclosed wild-type and altered variants of the TSC, NKCC2 and ROMK proteins as herein defined, preferably in isolated variant.
  • TSC TSC, NKCC2 or ROMK encoding nucleic acid molecules
  • the TSC, NKCC2 or ROMK genes or TSC, NKCCl or ROMK.
  • the nucleotide sequence of identified wild-type TSC encoding nucleic acid molecules are provided in Figure 2 and Table 3.
  • the nucleotide sequence of identified altered TSC encoding nucleic acid molecules are provided in Figures 2 and 4 and Tables 1 and 4.
  • the nucleotide sequence of identified wild-type NKCC2 encoding nucleic acid molecules are provided in Figure 7.
  • the nucleotide sequence of identified altered NKCC2 encoding nucleic acid molecules are provided in Figure 8 and Table 7.
  • the nucleotide sequence of identified altered ROMK encoding nucleic acid molecules are provided in Figure 11 and Table 10.
  • nucleic acid molecule is defined as an RNA or DNA molecule that encodes a peptide as defined above, or is complementary to a nucleic acid sequence encoding such peptides.
  • Particularly preferred nucleic acid molecules will have a nucleotide sequence identical to or complementary to the human cDNA sequences herein disclosed. Specifically contemplated are genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on an alternative backbone or including alternative bases whether derived from natural sources or synthesized.
  • nucleic acid molecules are defined further as being novel and unobvious over any prior art nucleic acid molecules encoding non-human homologues of TSC, NKCC2 or ROMK isolated from non-human organisms and known human ROMK proteins.
  • nucleic acid molecule is said to be "isolated” when the nucleic acid molecule is substantially separated from contaminant nucleic acid encoding other polypeptides.
  • a skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated TSC, NKCC2 or ROMK encoding nucleic acid molecule.
  • the present invention further provides fragments of the TSC, NKCC2 or ROMK encoding nucleic acid molecules of the present invention.
  • a fragment of a TSC, NKCC2 or ROMK encoding nucleic acid molecule refers to a small portion of the entire protein encoding sequence. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the TSC, NKCC2 or ROMK protein, such an intracellular or extracellular domain, then the fragment will need to be large enough to encode the functional region(s) of the TSC, NKCC2 or ROMK protein.
  • the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming.
  • Figures 2, 7, 11 and Tables 1-4, 6, 7, 9 and 10 identify fragments of the TSC, NKCCl and ROMK genes that are particularly useful as selective hybridization probes or PCR primers. Such fragments contain regions that are conserved among wild-type or altered variants of TSC, NKCC2 or ROMK, regions of homology that are shared with the previously identified TSC, NKCCl and ROMK genes, and regions that are altered in altered variants of the TSC, NKCCl and RDMK genes.
  • Fragments of the TSC, NKCC2 or ROMK encoding nucleic acid molecules of the present invention i.e., synthetic oligonucleotides
  • PCR polymerase chain reaction
  • Fragments of the TSC, NKCC2 or ROMK encoding nucleic acid molecules of the present invention can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al, J Am Chem Soc (1981) 103:3185-3191 or using automated synthesis methods.
  • DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the TSC, NKCCl or ROMK gene, followed by ligation of oligonucleotides to build the complete modified TSC, NKCCl or ROMK gene.
  • the TSC, NKCC2 or ROMK encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe purposes.
  • probes can be used to identify nucleic acid molecules encoding other allelic variants of wild-type or altered TSC, NKCC2 and ROMK proteins and as described below, such probes can be used to diagnosis the presence of an altered variant of a TSC, NKCC2 or ROMK protein as a means for diagnosing a pathological condition caused by abnormal ion transport.
  • a variety of such labels are known in the art and can readily be employed with the TSC, NKCC2 or ROMK encoding molecules herein described.
  • Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides, biotin, and the like. A skilled artisan can employ any of the art known labels to obtain a labeled TSC, NKCC2 or ROMK encoding nucleic acid molecule.
  • the identification of the role of the TSC, NKCC2 and ROMK proteins in the pathology/severity of ion transport mediated deficiencies has made possible the identification of several allelic variants of the wild-type TSC, NKCC2 and ROMK proteins as well as several altered variants of the TSC, NKCC2 and ROMK proteins that confer a pathology associated with abnormal ion transport.
  • a skilled artisan can readily use the amino acid sequence of the human TSC, NKCC2 and ROMK proteins to generate antibody probes to screen expression libraries prepared from cells.
  • polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a human cDNA or genomic expression library, such as lambda gtll library, prepared from a normal or effected individual, to obtain the appropriate coding sequence for wild-type or altered variants of the TSC, NKCC2 or ROMK protein.
  • the cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme.
  • Figures 2, 7 and 11 and Tables 1-4, 7, 9 and 10 identify important operative domains and domains that have been shown to contain alterations in mutated variants of each of the TSC, NKCC2 and ROMK proteins. Such regions are preferred sources of antigenic portions of the TSC, NKCC2 or ROMK protein for the production of probe, diagnostic, and therapeutic antibodies.
  • a portion of the TSC, NKCC2 or ROMK encoding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the TSC, NKCC2 or ROMK family of proteins from individuals that have normal ion transport or from individuals suffering from a pathological condition that is a result of abnormal ion transport.
  • Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives. This method can be used to identify and isolate altered and wild-type variants of the TSC, NKCC2 and ROMK encoding sequences.
  • pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively amplify/clone a TSC, NKCC2 or ROMK-encoding nucleic acid molecule, or fragment thereof.
  • PCR polymerase chain reaction
  • a PCR denature/anneal/extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other TSC, NKCC2 or ROMK encoding nucleic acid molecules.
  • Figures 2, 7 and 11 and Tables 1-4, 6, 7, 9 and 10 identify regions of the human TSC, NKCCl and ROMK genes that are particularly well suited for use as a probe or as primers.
  • the preferred primers will flank one or more exons of the TSC, NKCC2 or ROMK encoding nucleic acid molecule.
  • the present invention further provides methods for identifying cells and individuals expressing active and altered variants of the Na-K-2C1 cotransporter
  • NKCC2 the renal thiazide-sensitive Na-Cl cotransporter, TSC, and the ATP-sensitive potassium channel, ROMK.
  • Such methods can be used to diagnose biological and pathological processes associated with altered ion transport, particularly various variants of Bartter's Syndrome and Gitelman's Syndrome, the progression of such conditions, the susceptibility of such conditions to treatment and the effectiveness of treatment for such conditions.
  • the methods of the present invention are particularly useful in identifying carriers of ion transport deficiencies, particularly Gitelman's and Bartter's Syndromes, as well as in differentiating between Gitelman's and Banter's Syndromes.
  • the presence of wild-type or altered variants of the TSC, NKCC2 and ROMK proteins can be identified by determining whether a wild-type or altered variant of the TSC, NKCC2 or ROMK protein, or nucleic acid encoding one or more of these proteins, is expressed in a cell.
  • the expression of an altered variant, or departure from the normal level of TSC, NKCC2 or ROMK expression can be used as a means for diagnosing pathological conditions mediated by abnormal TSC, NKCC2 or ROMK activity/expression, differentiating between various ion transport deficiencies, and to identify carriers of ion transport deficiencies.
  • a variety of immunological and molecular genetic techniques can be used to determine if a wild-type or an altered variant of a TSC, NKCC2 or ROMK protein is expressed/produced in a particular cell and/or the level at which the protein is expressed. The preferred methods will identify whether a wild-type or mutated from of the TSC, NKCC2 or ROMK protein is expressed.
  • an extract containing nucleic acid molecules or an extract containing proteins is prepared from cells of an individual. The extract is then assayed to determine whether a TSC, NKCC2 or ROMK protein, or a TSC, NKCC2 or ROMK encoding nucleic acid molecule, is produced in the cell.
  • the type of protein/nucleic acid molecule expressed or the degree/level of expression provides a measurement of the nature and degree of TSC, NKCC2 or ROMK activity.
  • a suitable nucleic acid sample is obtained and prepared from a subject using conventional techniques. DNA can be prepared, for example, simply by boiling the sample in SDS.
  • a blood sample, a buccal swab, a hair follicle preparation or a nasal aspirate is used as a source of cells to provide the nucleic acid molecules.
  • the extracted nucleic acid can then be subjected to amplification, for example by using the polymerase chain reaction (PCR) according to standard procedures, to selectively amplify a TSC, NKCC2 or ROMK encoding nucleic acid molecule or fragment thereof.
  • PCR polymerase chain reaction
  • the size of the amplified fragment (typically following restriction endonuclease digestion) is then determined using gel electrophoresis or the nucleotide sequence of the fragment is determined (for example, see Weber and May Am J Hum Genet (1989) 44:388-339; Davies, J. et al. Nature (1994) 371 :130-136)).
  • the resulting size of the fragment or sequence is then compared to the known wild-type, predicted wild-type, known altered variants and predicted altered variants of the protein in question. Using this method, the presence of wild-type or altered variants of the TSC, NKCC2 and ROMK proteins can be differentiated and identified.
  • the presence or absence of one or more single base-pair polymorphism(s) within the TSC, NKCC2 or ROMK encoding nucleic acid molecules can be determined by conventional methods which included, but are not limited to, manual and automated fluorescent DNA sequencing, selective hybridization probes, primer extension methods (Nikiforov, T.T. et al. Nucl Acids Res (1994) 22:4167- 4175); oligonucleotide ligation assay (OLA) (Nickerson, D.A. et al. Proc Natl Acad Sci USA (1990) 87:8923-8927); allele-specific PCR methods (Rust, S.
  • a suitable protein sample is obtained and prepared from a subject using conventional techniques. Protein samples can be prepared, for example, simply by mixing the sample with SDS followed by salt precipitation of a protein fraction.
  • a blood sample, a buccal swab, a nasal aspirate, or a biopsy of cells from tissues expressing a TSC, NKCC2 or ROMK protein is used as a source of cells to provide the protein molecules.
  • the extracted protein can then be analyzed to determine the presence of a wild-type or altered variant of a TSC, NKCC2 or ROMK protein using known methods. For example, the presence of specific sized or charged variants of a protein can be identified using mobility in an electric filed. Alternatively, wild-type or altered variant specific antibodies can be used.
  • a skilled artisan can readily adapt known protein analytical methods to determine if a sample contains a wild-type or altered variant of a TSC, NKCC2 or ROMK protein.
  • TSC, NKCC2 or ROMK expression can also be used in methods to identify disorders that occur as a result of an increase or decrease in the expression of a naturally occurring TSC, NKCC2 or ROMK gene.
  • nucleic acid probes that detect mRNA can be used to detect cells or tissues that express a TSC, NKCC2 or ROMK protein and the level of such expression.
  • the presence of only an altered variant of a TSC protein (homozygous state) in a sample is diagnostic of Gitelman's Syndrome.
  • Altered variants of the TSC protein when present in sample that additionally contains a wild-type variant of TSC (heterozygous state), is diagnostic for carriers of Gitelman's Syndrome and individuals expressing lower levels of active TSC.
  • Decreased levels of active TSC lead to decreased urinary calcium, increased bone density and a propensity for deposition of calcium in the joints and diuretic induced hypokalaemia.
  • Elevated levels of TSC expression are diagnostic for increased urinary calcium, decreased bone density, and a propensity for high blood pressure and kidney stones.
  • NKCC2 protein The presence of only an altered variant of a NKCC2 protein (homozygous state) in a sample is diagnostic of several variants of Bartter's Syndrome.
  • Altered variants of the NKCC2 protein when present in sample that additionally contains a wild-type variant of NKCC2 (heterozygous state), is diagnostic for carriers of Bartter's Syndrome and individuals expressing lower levels of active NKCC2.
  • Decreased NKCC2 activity leads to increased urinary calcium, decreased bone mass and a propensity for kidney stones, osteoporosis and diuretic induced hypokalaemia.
  • the presence of only an altered variant of a ROMK protein (homozygous state) in a sample is diagnostic of several variants of Bartter's Syndrome.
  • Altered variants of the ROMK protein when present in sample that additionally contains a wild-type variant of ROMK (heterozygous state), is diagnostic for carriers of Bartter's Syndrome and individuals expressing lower levels of active ROMK. Decreased ROMK activity leads to increased urinary calcium, decreased bone mass and a propensity for kidney stones and osteoporosis.
  • TSC, NKCC2 or ROMK expression can also be used in methods to identify agents that increase or decrease the level of expression of a naturally occurring TSC, NKCC2 or ROMK gene.
  • cells or tissues expressing a TSC, NKCC2 or RMOK protein can be contacted with a test agent to determine the effects of the agent on TSC, NKCCl or ROMK expression.
  • Agents that activate TSC, NKCCl or ROMK expression can be used as an agonist of TSC, NKCC2 or ROMK activity whereas agents that decrease TSC, NKCCl or ROMK expression can be used as an antagonist of TSC, NKCC2 or ROMK activity.
  • the present invention further provides recombinant DNA molecules (rDNAs) that contain one or more of the wild-type or altered TSC, NKCC2 or ROMK encoding sequences herein described, or a fragment of the herein-described nucleic acid molecules.
  • rDNAs recombinant DNA molecules
  • an rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in vitro. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning (1989).
  • a TSC, NKCC2 or ROMK encoding DNA sequence that encodes a wild-type or altered variant of the TSC, NKCC2 or ROMK protein is operably linked to one or more expression control sequences and/or vector sequences.
  • the TSC, NKCC2 or ROMK encoding nucleic acid molecules will encode one of the novel altered or wild- type variants herein described.
  • a vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of a TSC, NKCC2 or ROMK encoding sequence included in the rDNA molecule.
  • Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, enhancers, transcription terminators and other regulatory elements.
  • inducible promoters that is readily controlled, such as being responsive to a nutrient in the host cell's medium, is used.
  • the vector containing a TSC, NKCC2 or ROMK encoding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule infrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule infrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic host cell such as a bacterial host cell, transformed therewith.
  • vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance.
  • Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycl
  • Vectors that include a prokaryotic replicon can further include a prokaryotic or viral promoter capable of directing the expression (transcription and translation) of the TSC, NKCC2 or ROMK encoding gene sequence in a bacterial host cell, such as E. coli.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, NJ.
  • Expression vectors compatible with eukaryotic cells can also be used to variant rDNA molecules that contain a TSC, NKCC2 or ROMK encoding sequence.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors.
  • Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. Southern et al, J Mol Anal Genet (1982) 1 :327-341.
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by cotransfection of the host cell, and selected by culturing in the presence of the appropriate drug for the selectable marker.
  • the present invention further provides host cells transformed with a nucleic acid molecule that encodes a human wild-type or altered TSC, NKCC2 or ROMK protein of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of a TSC, NKCC2 or ROMK protein are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the TSC, NKCC2 or ROMK gene product.
  • Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line, the most preferred being cells that do not naturally express a human TSC, NKCC2 or ROMK protein.
  • Any prokaryotic host can be used to express a TSC, NKCC2 or ROMK- encoding rDNA molecule.
  • the preferred prokaryotic host is E. coli. Transformation of appropriate cell hosts with an rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al, Proc Acad Sci USA (1972) 69:2110; and Maniatis et al, Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
  • the present invention further provides methods for producing a human wild- type or altered TSC, NKCC2 or ROMK protein that uses one of the TSC, NKCC2 or ROMK encoding nucleic acid molecules herein described.
  • the production of a recombinant human wild-type or altered TSC, NKCC2 or ROMK protein typically involves the following steps.
  • a nucleic acid molecule is obtained that encodes a TSC, NKCC2 or ROMK protein, such as the nucleic acid molecule depicted in Figures 2 and 7. If the TSC, NKCC2 or ROMK encoding sequence is uninterrupted by introns, it is directly suitable for expression in any host. If not, then a spliced variant of the TSC, NKCC2 or ROMK encoding nucleic acid molecule can be generated and used or the intron containing nucleic acid molecule can be used in a compatible eukaryotic expression system.
  • the TSC, NKCC2 or ROMK encoding nucleic acid molecule is then preferably placed in an operable linkage with suitable control sequences, as described above, to variant an expression unit containing the TSC, NKCC2 or ROMK encoding sequence.
  • the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the TSC, NKCC2 or ROMK protein.
  • the TSC, NKCC2 or ROMK protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • the desired coding sequences may be obtained from genomic fragments and used directly in an appropriate host.
  • the construction of expression vectors that are operable in a variety of hosts is accomplished using an appropriate combination of replicons and control sequences.
  • the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
  • Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • a skilled artisan can readily adapt any host/expression system known in the art for use with TSC, NKCC2 or ROMK encoding sequences to produce a TSC, NKCC2 or ROMK protein. Particularly well suited are expression systems that result in the production of lipid vesicles containing the expressed protein. Such lipid containing vesicles are well suited for identifying agonists and antagonists of the TSC, NKCC2 or ROMK protein.
  • I. Ion Transport As provided above, alterations in the TSC, NKCC2 or ROMK protein cause pathological conditions that are a result of abnormal ion transport. Accordingly, the wild-type and altered variants of the TSC, NKCC2 and ROMK proteins of the present invention can be used in methods to alter the extra or intracellular concentration of Na, Ca, Cl, Mg, and/or K. In general, the extra or intracellular concentration of Na, Ca, Cl, Mg, and/or K can be altered by altering the expression of a TSC, NKCC2 or ROMK protein or the activity of a TSC, NKCC2 or ROMK protein.
  • a TSC, NKCC2 or ROMK protein or TSC, NKCCl or ROMK gene expression can be used as a target for, or as means to alter extra or intracellular Na, Ca, Cl, Mg and/or K concentration.
  • a TSC, NKCCl or ROMK gene can be introduced and expressed in cells to increase TSC, NKCC2 or ROMK expression. This provides a means and methods for altering extra and intracellular ion levels.
  • TSC NKCC2 or ROMK activity/expression is targeted as a means of treating these conditions.
  • Various methods for regulating TSC, NKCC2 or ROMK activity/expression are discussed in detail below.
  • Another embodiment of the present invention provides methods for identifying agents that are agonists or antagonists of the TSC, NKCC2 and ROMK proteins herein described.
  • agonists and antagonists of a TSC, NKCC2 or ROMK protein can be first identified by the ability of the agent to bind to one of the wild-type or altered variants of the TSC, NKCC2 and ROMK proteins herein described.
  • Agents that bind to a TSC, NKCC2 or ROMK protein can then be tested for the ability to stimulate or block ion transport in a TSC, NKCCl or ROMK expressing cell.
  • a TSC, NKCC2 or ROMK protein is mixed with an agent. After mixing under conditions that allow association of TSC, NKCC2 or ROMK with the agent, the mixture is analyzed to determine if the agent bound the TSC, NKCC2 or ROMK protein. Agonists and antagonists are identified as being able to bind to a TSC, NKCC2 or ROMK protein.
  • the TSC, NKCC2 or ROMK protein used in the above assay can either be an isolated and fully characterized protein, can be a partially purified protein, can be a cell that has been altered to express a TSC, NKCC2 or ROMK protein or can be a fraction of a cell that has been altered to express a TSC, NKCC2 or ROMK protein. Further, the TSC, NKCC2 or ROMK protein can be the entire TSC, NKCC2 or
  • ROMK protein or a specific fragment of the TSC, NKCC2 or ROMK protein. It will be apparent to one of ordinary skill in the art that so long as the TSC, NKCC2 or ROMK protein can be assayed for agent binding, e.g., by a shift in molecular weight or change in cellular ion content, the present assay can be used.
  • the method used to identify whether an agent binds to a TSC, NKCC2 or ROMK protein will be based primarily on the nature of the TSC, NKCC2 or ROMK protein used.
  • a gel retardation assay can be used to determine whether an agent binds to a soluble fragment of a TSC, NKCC2 or ROMK protein whereas patch clamping, voltage clamping, ion-sensitive microprobes or ion-sensitive chromaphores can be used to determine whether an agent binds to a cell expressing a TSC, NKCC2 or ROMK protein and affects the activity of the expressed protein.
  • a skilled artisan can readily employ numerous techniques for determining whether a particular agent binds to a TSC, NKCC2 or ROMK protein.
  • the agent can be further tested for the ability to modulate the activity of a wild-type or altered variant of the TSC, NKCC2 or ROMK protein using a cell or oocyte expression system and an assay that detects TSC, NKCC2 or ROMK activity.
  • a cell or oocyte expression system and an assay that detects TSC, NKCC2 or ROMK activity.
  • voltage or patch clamping, ion-sensitive microprobes or ion-sensitive chromaphores and expression in Xenopus oocytes or recombinant host cells can be used to determine whether an agent that binds a TSC, NKCC2 or ROMK protein can agonize or antagonize TSC, NKCC2 or ROMK activity.
  • an agent is said to antagonize TSC, NKCC2 or ROMK activity when the agent reduces TSC, NKCC2 or ROMK activity.
  • the preferred antagonist will selectively antagonize TSC, NKCC2 or ROMK, not affecting any other cellular proteins, particularly other ion transport proteins. Further, the preferred antagonist will reduce TSC, NKCC2 or ROMK activity by more than 50%, more preferably by more than 90%, most preferably eliminating all TSC, NKCC2 or ROMK activity.
  • an agent is said to agonize TSC, NKCC2 or ROMK activity when the agent increases TSC, NKCC2 or ROMK activity.
  • the prefe ⁇ ed agonist will selectively agonize altered variants of TSC, NKCC2 or ROMK, not affecting any other cellular proteins, particularly other ion transport proteins. Further, the preferred agonist will increase TSC, NKCC2 or ROMK activity by more than 50%>, more preferably by more than 90%, most preferably more than doubling the level of TSC, NKCC2 or ROMK activity.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences of the TSC, NKCC2 or ROMK protein.
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the TSC, NKCC2 or ROMK protein.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to a fragment of a TSC, NKCC2 or ROMK protein.
  • agents of the present invention can be, as examples, peptides, small molecules, and vitamin derivatives, as well as carbohydrates.
  • a skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • One class of agents of the present invention are peptide agents whose amino acid sequences are chosen based on the amino acid sequence of the TSC, NKCC2 or ROMK protein.
  • the peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.
  • Another class of agents of the present invention are antibodies immunoreactive with critical positions of the TSC, NKCC2 or ROMK protein.
  • antibodies are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the TSC, NKCC2 or ROMK protein intended to be targeted by the antibodies.
  • Critical regions include the domains identified in Figures 5, 7 and 12.
  • TSC TSC
  • NKCC2 NKCC2
  • ROMK ROMK protein
  • the TSC, NKCC2 and ROMK proteins are involved in regulating intracellular and extracellular ion concentration.
  • Agents that bind a TSC, NKCC2 or ROMK protein and act as an agonist or antagonist can be used to modulate biological and pathologic processes associated with TSC, NKCC2 or ROMK function and activity.
  • a biological or pathological process mediated by TSC, NKCC2 or ROMK can be modulated by administering to a subject an agent that binds to a TSC, NKCC2 or ROMK protein and acts as an agonist or antagonist of TSC, NKCC2 or ROMK activity.
  • a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by TSC, NKCC2 or ROMK.
  • mammal means an individual belonging to the class
  • the invention is particularly useful in the treatment of human subjects.
  • a biological or pathological process mediated by TSC, NKCC2 or ROMK refers to the wide variety of cellular events mediated by a TSC, NKCC2 or ROMK protein.
  • Pathological processes refer to a category of biological processes which produce a deleterious effect.
  • pathological processes mediated by TSC, NKCC2 or ROMK include hypokalaemic alkalosis, hypokalaemic alkalosis with hypercalciuria, kidney stones, high blood pressure, osteoporosis and sensitivity to diuretic-induced hyperkalaemia.
  • These pathological processes can be modulated using agents that reduce or increase the activity of a TSC, NKCC2 or ROMK protein.
  • the agent will act to activate an otherwise inactive altered variant of a TSC, NKCC2 or ROMK protein.
  • an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process.
  • an agent is said to modulate Bartter's Syndrome when the agent contributes to normal intra and extracellular ion concentrations.
  • Agonists and Antagonists of a TSC, NKCC2 or ROMK Protein can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • an agent that modulates TSC, NKCC2 or ROMK activity is administered systemically or locally to the individual being treated. As described below, there are many methods that can readily be adapted to administer such agents.
  • the present invention further provides compositions containing an antagonist or agonist of a TSC, NKCC2 or ROMK protein that is identified by the methods herein described. While individual needs vary, a determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt.
  • the preferred dosages comprise 0.1 to 10 ⁇ g/kg ⁇ g/kg body wt.
  • the most preferred dosages comprise 0.1 to 1 ⁇ g/kg body wt.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble variant, for example, water-soluble salts.
  • suspensions of the active compounds and as appropriate, oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dintran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release variants thereof.
  • the agents of the present invention that modulate TSC, NKCC2 or ROMK activity can be provided alone, or in combination with another agents that modulate a particular biological or pathological process.
  • an agent of the present invention that reduces TSC, NKCC2 or ROMK activity can be administered in combination with other agents that affect the target ion transporter, for example, a diuretic agent.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time. - N. Animal Models and Gene Therapy
  • TSC, NKCCl and ROMK genes and the TSC, NKCC2 and ROMK proteins can also serve as targets for gene therapy in a variety of contexts.
  • TSC, NKCC2 or ROMK-deficient non-human animals can be generated using standard knock-out procedures to inactivate a TSC, NKCCl or ROMK gene or, if such animals are non- viable, inducible TSC, NKCC2 or ROMK antisense molecules can be used to regulate TSC, NKCC2 or ROMK activity/expression.
  • an animal can be altered so as to contain a TSC, NKCC2 or ROMK or antisense-TSC, NKCC2 or ROMK expression unit that directs the expression of TSC, NKCC2 or ROMK or the antisense molecule in a tissue specific fashion.
  • a non-human mammal for example a mouse or a rat, is generated in which the expression of the TSC, NKCCl or ROMK gene is altered by inactivation or activation. This can be accomplished using a variety of art-known procedures such as targeted recombination.
  • the TSC, NKCC2 or ROMK-deficient animal the animal that expresses TSC, NKCC2 or ROMK in a tissue specific manner, or an animal that expresses an antisense molecule can be used to 1) identify biological and pathological processes mediated by TSC, NKCC2 or ROMK, 2) identify proteins and other genes that interact with TSC, NKCC2 or ROMK, 3) identify agents that can be exogenously supplied to overcome TSC, NKCC2 or ROMK deficiency and 4) serve as an appropriate screen for identifying mutations within TSC, NKCCl or ROMK that increase or decrease activity.
  • transgenic mice expressing the human minigene for TSC, NKCC2 or ROMK in a tissue specific-fashion and test the effect of over-expression of the protein in cells and tissues that normally do not contain TSC, NKCC2 or ROMK.
  • This strategy has been successfully used for other proteins, namely bcl-1 (Veis et al Cell 75:229 (1993)).
  • Such an approach can readily be applied to the TSC, NKCC2 or ROMK protein and can be used to address the issue of a potential beneficial effect of TSC, NKCC2 or ROMK in a specific tissue area.
  • genetic therapy can be used as a means for modulating a TSC, NKCC2 or ROMK-mediated biological or pathological processes.
  • a genetic expression unit that encodes a modulator of TSC, NKCC2 or ROMK expression, such as an antisense encoding nucleic acid molecule or a TSC, NKCC2 or ROMK encoding nucleic acid molecule, or a functional TSC, NKCC2 or ROMK expression unit.
  • modulators can either be constitutively produced or inducible within a cell or specific target cell. This allows a continual or inducible supply of a modulator of TSC, NKCC2 or ROMK or the protein expression within a subject.
  • the resulting cDNA segments were radiolabeled (Feinberg, A.P., et al, Anal. Biochem. 132:6-13 (1983)) and used to screen a human genomic cosmid library by hybridization; the library and screening procedures have been described previously (Shimkets, R.A., et al, Cell 79:407-414 (1994)). Maps of resulting clones were defined by the overlapping products of digestion as well as by hybridization to specific exon or cDNA segments.
  • Genomic fragments bearing exons of TSC were subcloned by digestion of cosmid DNA to completion with Sau3AI and ligation of products into BamHI-digested pBluescript; resultant clones hybridizing to mouse or human TSC cDNA were isolated and subjected to DNA sequence analysis by the dideoxy chain termination method using an ABI 373 instrument and following a standard protocol.
  • primers flanking this simple sequence repeat were designed and used to direct PCR from genomic DNA of unrelated subjects (primer hTSCGT14-A: 5'-GTGAGCCACTGCGCTTAGCTG-3 * ; hTSCGT14-B: 5'-CTGCTGAGCTCTGGTCTGGAG-3'). Genotyping of CEPH and disease kindreds for loci indicated in the text was performed by polymerase chain reaction using specific primers (Gyapay, G., et al, Nature Genet.
  • Gitelman's Syndrome was prospectively distinguished from Bartter's Syndrome following the criteria of Bettinelli (Bettinelli, A., et al, J. Pediatr. 120:38- 43 (1992)): index cases in all of these families had marked hypocalciuria ( ⁇ 2 mg/kg/day, nl >4 mg/kg/day), hypomagnesemia ( ⁇ 0.5 meq/1, nl 0.8-1.0 meq/1), and symptomatic presentation after age 8. Twenty-seven patients were symptomatic, presenting with a variety of muscular symptoms including persistent muscular weakness, recurrent cramping with exertion, carpopedal spasm, total body tetany, and periodic paralysis with respiratory compromise.
  • TSCT-1 A polymo ⁇ hic genetic marker (TSCGT-1) at this locus was found by identification of a GT dinucleotide repeat sequence in the contig. Amplification of this segment from genomic DNA of unrelated subjects using specific primers (see Methods) demonstrated that this marker is polymo ⁇ hic, displaying 3 alleles and showing a heterozygosity of 48% in 45 unrelated Caucasian subjects.
  • This marker was shown to be linked to the TSC locus by finding this same marker on 4 independent cosmids from the cloned contig, as well as by subsequent linkage analysis of TSC variants with this marker in disease families (data not shown). This marker was used to localize TSC on the human genetic map by linkage in
  • CEPH pedigrees Pairwise analysis revealed strong evidence for linkage to a cluster of markers on human chromosome 16. Multipoint linkage analysis confirmed linkage to chromosome 16, with a peak lod score of 10.1 in the 3 cM interval flanked by D16S408 and D16S494, with odds favoring location in this interval of 950 to 1 over the next most likely interval (Figure 3). These findings localize the TSC on the human genetic map and identify highly informative genetic markers spanning the TSC locus, permitting us to test for linkage between this segment of chromosome 16 and Gitelman's Syndrome.
  • the lod-1 support interval localizes the gene to a 7 cM segment that includes the location of the TSC gene ( Figure 3).
  • Two other variants alter splice site consensus sequences, changing the CAG 3 ' consensus splice sequence to CAT at the junction of intron 15 and exon 16 (variant #5 in GITl 02, Tables 1 and 4), and changing GT to TT at the junction of exon 24 and intron 24 (#13 in GIT105). Both of these splice site variants are highly unlikely to give rise to normal proteins.
  • One variant (#7 in GIT 108) deletes 3 base pairs, resulting in deletion of a serine residue at codon 561; this serine residue is a potential protein kinase C phosphorylation site in the cytoplasmic carboxy terminus of the protein ( Figure 2c).
  • one variant (#10 in GITl 12) introduces a premature termination codon, deleting the last 54 amino acids from the encoded protein.
  • Primers (5'-3') are all within introns with the exception of: hTSCexl A-reverse and hTSCexl B-forward, which lie within exon 1 ; hTSCex26-forward lies at the beginning of exon 26 and hTSCex26-reverse lies distal to the normal termination codon.
  • R421 is also inherited by an affected second cousin, family member 11, by descent from their common ancestor; this subject is also a compound heterozygote, having inherited a third independent variant, altering a consensus splice site variant (variant #5; Tables 1 and 4, Figure 1) from her father.
  • hypokalaemic alkalosis is a relatively common complication of diuretic therapy of largely unknown cause. Mutant TSC alleles could contribute to development of this complication by further augmenting delivery of sodium and chloride to the distal nephron. This effect might be most likely in patients taking loop diuretics such as furosemide, and these mutations could consequently predispose to a particular complication of pharmacologic therapy.
  • NKCC2 cDNA sequence were used to direct PCR using a human kidney cDNA library as template. The sequence of the corresponding human cDNA encoding NKCC2 was determined; sequences were determined from both DNA strands.
  • Genotyping of CEPH and disease kindreds for loci indicated in the text was performed by polymerase chain reaction using specific primers (Gyapay, G. et al, Nature Genet. 7:246-339 (1994)) as described previously (Shimkets, R.A. et al, Cell 79:407-414 (1994)), except that the products were labeled by inclusion of 1 ⁇ Ci of [I- P]dCTP in the reaction mixture. All genotypes were scored independently by two investigators who were blinded to affection status. Analysis of linkage was performed using the LINKAGE programs Lathrop, G.M. et al, Proc. Natl. Acad. Sci. (U.S.A.) 81:3443-3446 (1984)).
  • Bartter's Syndrome kindreds 4 kindreds with at least one subject diagnosed with Bartter's Syndrome were studied. One of these cases has been reported previously (Di Pietro, A. et al, Ped. Med. Chir. 13:279-280 (1991)). In three of these families, at least one affected subject is known to be the offspring of consanguineous union ( Figure 6). In addition, in kindred BAR156 two second cousins of an index case are also affected. Index cases (Table 5) were all born prematurely with documented polyhydramnios and birth weight below 2 kg, and presented in the neonatal period with severe dehydration associated with marked hypokalaemia and hype ⁇ eninemic hyperaldosteronism.
  • TSC thiazide-sensitive Na-Cl cotransporter
  • NKCC2 Characterization of human NKCCl.
  • the predominant variant of human NKCCl cDNA isolated from a human kidney cDNA library encodes a protein of 1099 amino acids that shows strong sequence similarity to NKCC2 cDNAs characterized from rabbit (Payne, J.A., et al, Proc. Natl. Acad. Sci. (U.S.A.) 91:4544-4548 (1994)) (95% amino acid identity), and rat (Gamba, G. et al, J. Biol. Chem. 26:17713-17722 (1994)) (93% identity).
  • human NKCC2 shows considerable similarity in amino acid sequence to NKCCl from shark rectal gland (Xu, J.-C. et al, Proc. Natl. Acad. ScL (U.S.A.)
  • Plasmid artificial chromosome (PAC) (Ioannou, P. A. et al, Nature Genet. 6:84-89 (1994)) clones containing the human genomic NKCC2 locus were isolated, and one of these, NKCC2-6A, was found to contain all the coding exons. This permitted determination of the intron-exon structure of the gene (see Methods).
  • the NKCC2 protein is encoded in 26 exons ( Figure 7a ); introns range in length from 120 base pairs to 15 kb, and the coding region spans a total of 80 kb in genomic DNA. The location of intron-exon boundaries is very similar to those seen in TSC through exon 19 (Simon, D.B.
  • NKCCl was localized to human chromosome 15 (data not shown).
  • a (GT) dinucleotide repeat sequence was identified at the cloned NKCCl locus; this repeat proved to be polymo ⁇ hic in genomic DNA, with 5 alleles and 42% heterozygosity in 50 unrelated subjects.
  • This marker was genotyped in CEPH reference kindreds in order to localize NKCCl on the human genetic map; analysis revealed linkage to a cluster of loci at 15q 15-21. Multipoint analysis yielded a maximum lod score of 13.3 for linkage to a 3 centimorgan interval flanked by D15S132 and D15S209 (Figure 7c); the odds favoring location in this interval were more than 100 times as likely as location in the next most likely interval (Figure 7c).
  • NKCCl Homozygosity of NKCCl in Bartter's kindreds.
  • all affected offspring of consanguineous union were homozygous for alleles of each marker, and these haplotypes cosegregated with the disease, providing strong evidence supporting linkage of Bartter's Syndrome and the NKCCl locus ( Figure 6).
  • the affected subject in BAR138 is not known to be the product of consanguineous union, she too is homozygous for all markers tested in this segment.
  • Her affected second cousins have inherited one copy of the same variant along with a portion of her haplotype; these subjects have presumably inherited a second, and as yet undetected, variant on the maternally inherited allele. None of these variants have been observed in examination of 80 alleles from unrelated subjects who do not have Bartter's Syndrome, and no variants altering the amino acid sequence have been detected in unrelated unaffected subjects.
  • the variant in BAR152 is a 1 base pair insertion introducing a frameshift mutation at codon Ml 95 in the first transmembrane domain.
  • the variant in BAR138 represents a 1 base pair deletion in codon R302 in the fourth transmembrane domain, also resulting in a frameshift mutation. Both of these mutations are extremely unlikely to yield proteins with normal function.
  • the other two variants result in non-conservative missense mutations at residues that are conserved among members of the NKCC family from distantly related species.
  • the variant in BAR156 substitutes phenylalanine for valine at residue 272 in the third transmembrane domain. This valine residue is conserved in NKCC2 from human, rabbit, mouse, and rat, as well as NKCCl in shark and human.
  • the variant in BAR165 substitutes an asparagine residue for aspartate at amino acid 648, just distal to the 12th transmembrane domain; this aspartate residue is also conserved in every member of the NKCC family.
  • K + serum potassium (mM, nl 3.5-5.0 mM); HC0 3 ' : serum bicarbonate ( M, nl 23-26 mM); UCa/UCr: urinary calcium.-creatinine ratio (mM:mM; nl ratio 0.2 - 0.4).
  • Nephrocalcinosis determined by ultrasonography. Preterm: birth before 36 weeks' gestation. ND, not done. Table 6.
  • Primers are all within introns with the exception of: hNKCC2ex 1 A-reverse and hNKCC2ex1 B-forward, which lie within exon 1 ; hNKCC2ex26-reverse lies distal to the normal termination codon.
  • Urinary prostaglandin E2 levels have been reported to be elevated in patients with Bartter's Syndrome (Seyberth, H.W. et al, Pediatr. Nephrol 1 :491-497 (1987); Gill, J.R., Jr., et al, Am. J. Med. 61:43-51 (1976)); high PGE2 levels can be produced by administration of loop diuretics (Dunn, M.J., Kidney Int. 18:86-102 (1981)), consistent with genetic loss of Na-K-2C1 function resulting in a secondary increase in PGE2 levels.
  • hypokalaemic alkalosis Classification of hypokalaemic alkalosis. It is now apparent that inherited hypokalaemic alkalosis is genetically heterogeneous and due to mutations in genes encoding at least two proteins — the Na-K-2C1 cotransporter of the thick ascending limb of the loop of Henle (NKCC2) and the Na-Cl cotransporter of the distal convoluted tubule (TSC).
  • NKCC2 the Na-K-2C1 cotransporter of the thick ascending limb of the loop of Henle
  • TSC distal convoluted tubule
  • hypokalaemic alkalosis with normal or low blood pressure will prove due to mutation in one of these two genes or whether further genetic heterogeneity will be found is unresolved.
  • a group of patients has been described with severe magnesium wasting, hypokalaemic alkalosis and nephrocalcinosis (Gullner Syndrome) (Gullner, H.-G., et al, Am. J. Med. 71 :578-582 (1981)).
  • the hypokalaemia in these patients has been reported to be corrected with magnesium replacement.
  • NKCCl and TSC are closely related, with similar highly conserved sequence, structure and function. From this, one might expect the prevalence of mutant alleles in these genes, and the prevalence of clinical disease, to be similar.
  • Gitelman's Syndrome defined by the presence of hypocalciuria
  • Bartter's Syndrome defined by the presence of hypercalciuria
  • mutant NKCCl alleles have severe effects when homozygous, they may also have detectable phenotypes in the more prevalent heterozygotes.
  • lowered blood pressure is a reasonable possibility, owing to reduced sodium reabso ⁇ tion in the thick ascending limb, leading to a reduced set point of sodium balance and decreased intravascular volume.
  • loop diuretics to lower blood pressure by inhibition o ⁇ NKCCl supports this possibility.
  • Another potential phenotype in heterozygotes is susceptibility to diuretic- induced hypokalaemia.
  • Thiazide diuretics are commonly used anti-hypertensive agents that inhibit TSC in the distal convoluted tubule; thiazide-induced hypokalaemia is a relatively common complication. Heterozygotes for NKCC2 mutations may be able to compensate for diminished sodium reabso ⁇ tion in the thick ascending limb by increased distal reabso ⁇ tion via TSC in the distal convoluted tubule (Ellison, D.H. Ann. Int. Med. 1 14:886-894 (1991)) (Figure 9).
  • NKCC2 mutations have marked urinary calcium wasting, leading to both early and severe nephrocalcinosis and marked demineralization of bone. Salt wasting in heterozygotes for NKCC2 mutations might also result in hypercalciuria, potentially increasing susceptibility to nephrolithiasis (renal stones) and osteoporosis (bone demineralization), multifactorial traits with known inherited components.
  • mutations in an X-linked renal chloride channel have been shown to promote development of nephrolithiasis (Lloyd, S.E. et al, Nature 379:445- 449 (1996)), consistent with the notion that primary abnormalities in renal sodium and chloride handling may underlie stone development in a significant fraction of affected subjects.
  • Genotyping and linkage analysis Genotyping of loci indicated in the text was performed by polymerase chain reaction using specific primers as described previously (Simon, D.B., et al, Nature Genet. 13: 183-188 (1996)). All genotypes were scored independently by two investigators who were blinded to affection status. Analysis of linkage was performed using the LINKAGE programs (Lathrop, G.M. et al, Proc. Natl. Acad. Sci. (U.S.A.) 81:3443-3446 (1984)), specifying Bartter's Syndrome as an autosomal recessive trait with 99% penetrance and a prevalence of phenocopies of 0.1%.
  • NKCCl and TSC were as described previously (Examples 1 and 2 and Simon, D.B. et al, Nature Genet. 12:24-30 (1996), Simon, D.B., et al, Nature Genet. 13:183-188 (1996)). Primer pairs were employed to direct PCR using genomic DNA of disease family members or controls as a template as previously described (Simon, D.B. et al, Nature Genet. 12:24-30 (1996)).
  • Affected subjects of BAR159 and BAR161 were screened for ROMK mutations by SSCP. Homozygous ROMK variants were found in affected subjects of both kindreds, and these variants cosegregated with the disease ( Figures 10 and 11a, b). These variants are not common in the population, as they are not detected on 80 chromosomes from unaffected unrelated subjects from Saudi Arabia and the CEPH reference pedigrees. By homozygosity mapping, specifying these variants as rare, the lod score for linkage of ROMK and Bartter's Syndrome in these two families is 3.2, supporting linkage.
  • This serine is highly conserved among members of the IRK family of K + channels and represents a protein kinase A phosphorylation site in the cytoplasmic carboxy terminus of the protein; this site has been shown to be phosphorylated by PKA, and substitution of alanine by site- directed mutagenesis has shown that this site is required for normal K + channel activity, with mutant ROMK showing an approximately 50% reduction in channel activity (Xu, Z-C, et al, J. Biol. Chem. 271 :9313-9319 (1996)).
  • the second variant in this kindred represents another premature termination codon, occurring at codon 58, again truncating the encoded protein prior to the first transmembrane domain ( Figure 12).
  • one variant represents a 4 base pair deletion, spanning the last base of codon T313 and all of codon K314, resulting in a frameshift mutation and altering the encoded protein from amino acid 315 onward, ending at a new stop codon at position 350 ( Figures 1 lc, 12).
  • the second variant in this kindred arises from substitution of valine for alanine at amino acid 195, in the cytoplasmic carboxy terminus of ROMK ( Figure 12); this alanine residue is conserved in rat and human ROMK, and has not been observed in unrelated unaffected subjects.
  • BAR139 in which no NKCC2 variants have been identified a single ROMK variant, substituting threonine for methionine at amino acid M338 was identified ( Figure 12); this variant has not been seen on 80 chromosomes from unaffected subjects. As yet, no mutation has been identified on the other ROMK allele in this affected subject.
  • K + serum potassium (mM, nl 3.5-5.0 mM); HC0 3 : serum bicarbonate (mM, nl 23-26 mM); UCa/UCr: urinary calcium:creatinine ratio (mM:mM, nl ⁇ 0.4 ).
  • Nephrocalcinosis determined by ultrasonography. Consang. : Offspring of consanguineous union. Table 9. PCR Primers for SSCP Analysis of the Human ROMK Gene
  • NKCCl and ROMK are the sole genes in which mutation causes Bartter's Syndrome, or whether additional genes will be identified remains an open question; of the total of 15 families harboring independent Bartter's mutations studied thus far (Simon, D.B., et al, Nature Genet. 13: 183-188 (1996) and the present study), NKCCl variants have been identified or implicated by linkage in 10 families, and ROMK mutations have been identified in 5 families. In one outbred family mutation in either ROMK or NKCC2 have not been found to date. This could reflect either incomplete sensitivity of mutation detection or alternatively could be accounted for by further genetic heterogeneity.
  • the pathophysiology seen in affected patients can be explained by the loss of ROMK function resulting in inability to recycle K + from cells of the TAL back into the renal tubule, resulting in K + levels in the lumen that are too low to permit continued Na-K-2C1 cotransport activity, producing salt wasting from this nephron segment.
  • This salt wasting results in activation of the renin-angiotensin system, increased aldosterone levels, and increased electrogenic sodium reabso ⁇ tion via the epithelial sodium channel of the distal nephron in exchange for K + and H , accounting for the observed hypokalaemic alkalosis. Since calcium reabso ⁇ tion is coupled to Na + reabso ⁇ tion in the TAL, loss of Na + transport in this segment leads to the characteristic hypercalciuria of these patients.
  • ROMK isoforms are also expressed at more distal sites in the nephron, and it has been proposed that isoforms of this same channel contribute to net renal potassium secretion in the distal nephron (Lee, W.S., et al, Am. J. Physiol (Renal Fluid Electrol. Physiol.) 268:F1124-31 (1995), Boim, M.A. et al, Am. J. Physiol. (Renal Fluid Electrol. Physiol.) 268:F1132-40 (1995), Hebert, S.C, Kidney Int. 48:1010-1016 (1995)).
  • ROMK isoforms do not play an indispensable role in distal renal potassium excretion in vivo in humans. Further work will be required to address this question and to determine whether the clinical features of patients with mutations in these two genes can be distinguished clinically. In addition, while ROMK isoforms are also expressed in brain, spleen, lung and eye (Hebert, S.C, Kidney Int. 48:1010-1016 (1995)), there are no obvious phenotypes in these patients that suggest an essential role for ROMK function in these organs.

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Abstract

La présente invention concerne, pour partie, l'identification des rôles du co-transporteur de Na-Cl, sensible aux composés thiazidiques, d'origine humaine, le TSC; du canal K+ sensible à l'ATP d'origine humaine, le ROMK; et du co-transporteur de Na-K-2Cl d'origine humaine, le NKCC2, s'agissant de la création d'un trouble pathologique associé à un transport d'ions anormal, notamment dans le syndrome de Bartter, le syndrome de Gitelman, l'alcalose hypokaliémique, l'alcalose hypokaliémique avec hypercalciurie, les calculs rénaux, l'hypertension artérielle, l'ostéoporose et la sensibilité à l'hyperkaliémie induite par diurétique. La présente invention se rapporte spécifiquement à la séquence d'acides aminés de plusieurs allèles modifiés ou du type sauvage, d'origine humaine, des protéines TSC, NKCC2 et ROMK ainsi qu'à la séquence nucléotidique qui code ces allèles pouvant servir au diagnostic des troubles liés au transport d'ions.
PCT/US1997/023553 1996-12-31 1997-12-19 Procede pour diagnostiquer et traiter des troubles pathologiques lies a une insuffisance du transport d'ions WO1998029431A1 (fr)

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AU60135/98A AU746220B2 (en) 1996-12-31 1997-12-19 Method to diagnose and treat pathological conditions resulting from deficient ion transport
EP97954793A EP1030858A1 (fr) 1996-12-31 1997-12-19 Procede pour diagnostiquer et traiter des troubles pathologiques lies a une insuffisance du transport d'ions
JP53012398A JP2001508291A (ja) 1996-12-31 1997-12-19 不充分なイオン輸送によって生じる病理学的状態の診断および処置のための方法
CA002274955A CA2274955A1 (fr) 1996-12-31 1997-12-19 Procede pour diagnostiquer et traiter des troubles pathologiques lies a une insuffisance du transport d'ions

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WO2000005372A1 (fr) * 1998-07-22 2000-02-03 Incyte Pharmaceuticals, Inc. Proteine humaine de transport d'ions
WO2002080842A2 (fr) * 2001-04-05 2002-10-17 Estetecon Ab Medicament et procede de diagnostic d'un etat auto-immun
US7183075B2 (en) 2000-04-14 2007-02-27 Vanderbilt University Purified and isolated potassium-chloride cotransporter nucleic acids and polypeptides and therapeutic and screening methods using same

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JP6586684B2 (ja) * 2015-11-11 2019-10-09 島根県 ウシ胎子の胎膜水腫の発症しやすさを判定する方法及びウシの胎膜水腫のキャリアを特定する方法

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AMERICAN JOURNAL OF HUMAN GENETICS, November 1996, Vol. 59, MASTROIANNI et al., "Novel Molecular Variants of the Na-Cl Cotransporter Gene are Responsible for Gitelman Syndrome", pages 1019-1026. *
AMERICAN JOURNAL OF PHYSIOLOGY, September 1995, Vol. 269, IGARASHI et al., "Cloning, Embryonic Expression and Alternative Splicing of a Murine Kidney-Specific Na-K-Cl Cotransporter", pages F405-F418. *
MOLECULAR PHARMACOLOGY, May 1994, Vol. 45, YANO et al., "Alternative Splicing of Human Inwardly Rectifying K+ Channel ROMK1 mRNA", pages 854-860. *
NATURE GENETICS, January 1996, Vol. 12, SIMON et al., "Gitelman's Variant of Bartter's Syndrome, Inherited Hypokalaemic Alkalosis, is Caused by Mutations in the Thiazide-Sensitive Na-Cl Cotransporter", pages 24-30. *
NATURE GENETICS, June 1996, Vol. 13, SIMON et al., "Bartter's Syndrome, Hypokalaemic Alkalosis with Hypercalciuria, is Caused by Mutations in the Na-K-2Cl Cotransporter NKCC2", pages 183-188. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000005372A1 (fr) * 1998-07-22 2000-02-03 Incyte Pharmaceuticals, Inc. Proteine humaine de transport d'ions
US7183075B2 (en) 2000-04-14 2007-02-27 Vanderbilt University Purified and isolated potassium-chloride cotransporter nucleic acids and polypeptides and therapeutic and screening methods using same
WO2002080842A2 (fr) * 2001-04-05 2002-10-17 Estetecon Ab Medicament et procede de diagnostic d'un etat auto-immun
WO2002080842A3 (fr) * 2001-04-05 2003-01-23 Estetecon Ab Medicament et procede de diagnostic d'un etat auto-immun

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EP1030858A1 (fr) 2000-08-30
CA2274955A1 (fr) 1998-07-09
JP2001508291A (ja) 2001-06-26
AU746220B2 (en) 2002-04-18

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