WO2010120746A1 - Polymorphisms in human trpv4 and methods of use thereof - Google Patents

Abstract

Compositions and methods for diagnosing aberrant water metabolism are disclosed.

Description

Polymorphisms in Human TRPV4 and Methods of Use Thereof

By David M. Cohen

Wei Tian

This application claims priority under 35 U. S. C.

§119 (e) to U.S. Provisional Patent Application No. 61/169,542, filed on April 15, 2009. The foregoing application is incorporated by reference herein.

This invention was made with government support under Grant No. R21 AG029968 awarded by the National Institutes of Health and under a Veterans Affairs Merit Review Grant entitled "Regulation of the hypotonicity sensor TRPV4" awarded by the Department of Veterans Affairs. The government has certain rights in the invention.

FIELD OF THE INVENTION The present invention relates to the field of aberrant water metabolism. Specifically, polymorphisms in transient receptor potential-vanilloid-4 (TRPV4) which are associated with hyponatremia are disclosed.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Systemic osmolality is among the most tightly regulated of physiological parameters. In humans, aberrant water balance is associated with neurological dysfunction and death. Even subtle changes in systemic osmolality cause reversible defects in coordination and cognition (Renneboog et al. (2006) Am. J. Med., 119:71 el-8; Schrier et al. (2006) N. Engl. J. Med., 355:2099- 112) . Clinically, water balance is reflected in the serum (or plasma) sodium concentration. Water excess relative to total body sodium content results in hyponatremia - the most prevalent electrolyte abnormality in hospitalized patients (Ellison et al. (2007) N. Engl. J. Med., 356:2064-72; Verbalis et al. (2007) Disorders of Water Balance. In: Brenner BM, ed. Brenner and Rector's The Kidney: Saunders) .

In mammals, systemic water balance is regulated via the renal water-conserving role of the hormone, arginine vasopressin. Release of arginine vasopressin from the posterior pituitary into the circulation is governed by the hypothalamic sensor (s) of systemic osmolality.

Abundant evidence suggests that the transient receptor potential channel TRPV4 comprises an element of the central sensor of low osmolality. TRPV4 is the mammalian ortholog of a C. elegans osmosensing protein (Liedtke et al. (2000) Cell, 103:525-35; Strotmann et al. (2000) Nat. Cell Biol., 2:695-702) . In rodents, the channel is expressed in the osmosensing nuclei of the brain (Liedtke et al. (2000) Cell, 103:525-35), among other sites. In vitro, TRPV4 is activated by hypotonicity (Liedtke et al. (2000) Cell, 103:525-35; Strotmann et al. (2000) Nat. Cell Biol., 2:695-702; Wissenbach et al. (2000) FEBS Lett., 485:127-34) and by a number of lipid agonists including phorbol ester derivatives (Watanabe et al. (2002) J. Biol. Chem. , 277:13569-77) . Osmotic and mechanical sensitivity of TRPV4 is ultimately conferred by the arachidonic acid metabolites and epoxyeicosatrienoic acids (EET) , following phospholipase A2 activation (Watanabe et al. (2003) Nature, 424:434-8; Vriens et al. (2004) Proc. Natl. Acad. Sci., 101:396-401; Andrade et al. (2005) J. Cell Biol., 168:859-874) . Other signaling pathways involving inositol trisphosphate (Fernandes et al. (2008) J. Gen. Physiol., 131:i2; Garcia-Elias et al. (2008) J. Biol. Chem., 283:31284-8), SRC-family tyrosine kinases (Xu et al. (2003) J. Biol. Chem., 278:11520-7; Wegierski et al. (2009) J. Biol. Chem., 284:2923-33), and sensitization by co-application of different stimuli (Gao et al. (2003) J. Biol. Chem., 278:27129-37; Alessandri-Haber et al. (2003) Neuron, 39:497-511) also impact the TRPV4 response to osmotic and mechanical stimulation. In vivo, TRPV4 participates in the regulation of arginine vasopressin release in mouse, where targeted deletion of the TRPV4 gene gives rise to aberrant systemic osmoregulation (Mizuno et al. (2003) Am. J. Physiol. Cell Physiol., 285 :C96-C1O1; Liedtke et al. (2003) Proc. Natl. Acad. Sci., 100:13698-703).

Exceedingly rare Mendelian defects in the genes coding for the kidney collecting duct-specific water channel (aquaporin-2; AQP2 gene), arginine vasopressin (AVP gene) , and arginine vasopressin receptor-2 (AVPR2 gene) cause profound water wasting (Bahnsen et al. (1992) Embo J., 11:19-23; Ito et al. (1991) J. Clin. Invest., 87:725-8; Deen et al. (1994) Science, 264:92-5; van den Ouweland et al. (1992) Nat. Genet., 2:99-102) or water retention (Feldman et al. (2005) N. Engl. J. Med., 352:1884-90), although without major repercussion at the population level. To date, no human mutation in an osmosensing TRP channel has been shown to impact osmoregulation, and no polymorphisms impacting systemic water balance have been reported for any gene. SUMMARY OF THE INVENTION

In accordance with the present invention, methods of diagnosing an increased risk for hyponatremia in a patient are provided. In a particular embodiment, the methods comprise assessing the presence of at least one hypofunctioning allele of TRPV4 in a biological sample from the patient. In yet another embodiment, the hypofunctioning allele comprises a mutation of the proline residue at position 19 of TRPV4 (e.g., a serine for the proline residue) • In still another embodiment, the TRPV4 allele comprises a mutation (e.g., a non- conservative change) of the alanine residue at position 10 of TRPV4 (e.g., a proline for the alanine residue).

BRIEF DESCRIPTIONS OF THE DRAWING

Figures 1A-1F demonstrate that the presence of the TRPV4^P19S allele is associated with hyponatremia. Prevalence of the TRPV4^P19s allele among genotyped non- Hispanic Caucasian (Cauc; Fig. IA) and African American (AA/ Fig. IB) subjects in the Healthy Aging cohort from the Layton Center for Aging and Alzheimer's Disease, and from the genotyped non-Hispanic Caucasian subjects in the Osteoporotic Fractures in Men Study (MrOS; Fig. 1C) , expressed as a function of serum sodium concentration. Depicted is the prevalence of the heterozygous genotype as percent of total number of subjects with serum sodium concentration at or below the indicated level. "All" denotes prevalence for the entire genotyped population. Figure ID provides a graph of the distribution of serum sodium concentration (binned as integers) for all

Caucasian subjects in the MrOS cohort with creatinine < 1.3 mg/dl and glucose < 150 mg/dl (n = 4409) . Prevalence of the TRPV4^pl9s polymorphism is shown in the inset bar graph, where Low corresponds to [Na⁺] ≤ 138 mEq (milliequivalent ) /1 (~ lowest decile) , Mean is [Na⁺] 141 or 142 mEq/1, and High represents [Na⁺] > 145 mEq/1 (~ highest decile) ; bars are keyed to the frequency distribution via shading. Figure IE is a graph demonstrating the prevalence of hyponatremia, defined as serum sodium concentration ≤ 135 mEq/1, among subjects with one ("P19S-positive") or no ("P19S-negative") TRPV4^P19S alleles in non-Hispanic Caucasian (Cauc; n = 219) and African American (AA; n = 72) subjects in the Healthy Aging cohort, and in non-Hispanic Caucasian MrOS subjects (n = 1300) . Figure IF is a graph depicting the mean serum sodium concentration (± SD) in the three cohorts, expressed as a function of the presence or absence of one TRPV4^P19S allele. Sodium concentration was significantly lower (by 0.9 - 2.4 mEq/1) in all three groups of TRPV4^pl9s-positive subjects relative to TRPV4^pl9s-negative subjects; p = 0.05, 0.014, and 0.04 via t-Test for the non-Hispanic Caucasian and African American Healthy Aging subjects and for the non-Hispanic Caucasian MrOS cohort, respectively.

Figures 2A-2G provide graphs demonstrating that the TRPV4^P19S allele is hypofunctioning in vitro. Time courses for whole-cell currents at -100 mV and +100 mV in HEK293 cells transfected with human TRPV4^WT (Fig. 2A) and TRPV4^P19S (Fig. 2B) exposed to 15% hypotonicity and then 4α-phorbol 12, 13-didecanoate (4αPDD; 10 mM) . Figs. 2C and 2D depict the corresponding whole-cell current/voltage relationships recorded at the times indicated by the color-coded boxes in Figs. 2A and 2B, respectively. Fig. 2E represents the mean current density (expressed as pA/pF at +100 mV) of TRPV4^WT and TRPV4^P19S upon exposure to mild (i.e., 15%; left) and more pronounced (i.e., 30%; right) hypotonicity. Fig. 2F shows representative current/voltage relationship at peak (maximum) currents from TRPV4^WT and TRPV4^P19S transfectants loaded with 156 nM epoxyeicosatrienoic acid (EET) in the pipette solution, and the mean responses (Fig. 2G) . Data are expressed as the mean ± SEM of the number of experiments shown in brackets. Statistical significance, determined via t-test, is indicated.

Figure 3 provides images showing that TRPV4^WT and TRPV4^P19S are expressed to an equivalent degree at the plasma membrane in transfected HEK cells. HEK cells transfected with the wild-type TRPV4 (Upper) or the P19S variant (Lower) as in Figure 2 were subjected to confocal immunofluorescence microscopy (Right) or brightfield microscopy (Left) following immunolabeling for TRPV4. Abundant staining of the plasma membrane is noted in both transfectants. Scale bar: 10 μm.

Figure 4 provides an image of a gel demonstrating the equivalent cell-surface expression of TRPV4 and variant TRPV4 following transient transfection in HEK293 cells. Cells were transiently transfected with full- length cDNA coding for wild-type human TRPV4 (hTRPV4) or for human TRPV4 point-mutated to incorporate either the A565T or the P19S nonsynonymous polymorphisms. TRPV4 expression in whole-cell lysates was equivalent in the 3 transfectants (right half of the figure) in the depicted anti-TRPV4 immunoblot. Cell surface expression (left half of the figure) was similarly equivalent, as demonstrated via avidin-affinity precipitates following cell surface biotinylation (see Methods) . Molecular mass markers (in kDa) are shown on the left; arrowheads indicate the TRPV4.

Figure 5 shows the immunodetection of TRPV4 in transfected HEK293 cells. HEK293 cells transfected with TRPV4 + GFP (Top) or GFP alone (Bottom) . (Left) Transmitted light images. (Center) GFP fluorescence. (Right) TRPV4 immunofluorescence signal.

Figure 6 provides a graph of the whole-cell currents recorded in HEK293 cells transfected with GFP and exposed to 30% hypotonic shock. Inset shows average current density. P > 0.05 Student's t test.

DETAILED DESCRIPTION OF THE INVENTION Disorders of water balance are among the most common and morbid of the electrolyte disturbances, and are reflected clinically as abnormalities in the serum sodium concentration. The transient receptor potential- vanilloid-4 (TRPV4) channel is postulated to comprise an element of the central hypotonicity-sensing mechanism in the mammalian hypothalamus, and is activated by hypotonic stress in vitro. The TRPV4 gene was described as the mammalian ortholog (Liedtke et al. (2000) Cell 103:525-535; Strotmann et al. (2000) Nat. Cell Biol., 2:695-702) of a worm gene (Colbert et al. (1995) Neuron, 14:803-812; Colbert et al. (1997) J. Neurosci., 17:8259- 8269) , the product of which was believed to be responsive to changes in salt concentration. The gene codes for a channel protein that admits calcium and other ions into the cell only in response to specific activating stimuli. When TRPV4 is expressed in cells that do not normally make the protein, it is activated by dilution of the medium bathing cells (Liedtke et al. (2000) Cell 103:525-535; Strotmann et al. (2000) Nat. Cell Biol., 2:695-702) . TRPV4 is expressed in regions of the brain that are responsible for monitoring the concentration (i.e., water content) of the blood (Liedtke et al. (2000) Cell 103:525-535; Strotmann et al. (2000) Nat. Cell Biol., 2:695-702) . It is also found in regions of the kidney where water excretion is tightly regulated (Tian et al. (2004) Am. J. Physiol. Renal Physiol., 287:F17-24) . Mice that have had their TRPV4 gene "knocked out" exhibit abnormal water balance (Liedtke et al. (2003) Proc. Natl. Acad. Sci.,

100:13698-13703; Mizuno et al. (2003) Am. J. Physiol. Cell Physiol., 85:C96-C1O1) . Accordingly, TRPV4 is pivotal for water balance.

As described herein, a nonsynonymous single nucleotide polymorphism (a OT change (i.e., the CCC codon for proline to TCC codon for serine) in the TRPV4 gene gives rise to a Pro-to-Ser substitution at residue 19. Association of this human polymorphism with serum sodium concentration and with hyponatremia (serum sodium concentration ≤ 135 mEq/1; relative water excess) was tested in the Healthy Aging cohort of the Layton Aging and Alzheimer's Disease Center at Oregon Health & Science University. Confirmation of association was sought using banked serum and genomic DNA from the Osteoporotic Fractures in Men (MrOS) Study. Function of the variant TRPV4^P19S allele was tested in vitro in heterologously transfected human embryonal kidney (HEK293) cells.

The TRPV4^P19S polymorphism was significantly associated with serum sodium concentration and with the presence of hyponatremia in both non-Hispanic Caucasian male populations; in addition, mean serum sodium concentration was significantly lower (by 0.9 - 2.4 mEq/1) among subjects with one copy of the TRPV4^pl9s allele relative to the wild-type allele. No other functional polymorphisms in linkage disequilibrium with rs3742030 were identified. Subjects with the minor allele were 6.45 or 2.43 times (depending upon cohort) as likely to exhibit hyponatremia as subjects without the minor allele after inclusion of key covariates. Consistent with these observations, a human TRPV4 channel mutated to incorporate the TRPV4^P19S polymorphism showed diminished response to mild hypotonic stress and to the osmotransducing lipid messenger epoxyeicosatrienoic acid, but not to more pronounced hypotonic stress or to a synthetic channel agonist (4αPDD) , in heterologous expression studies. In addition, insofar as oligomerization is required forTRPV4channel function (Arniges et al. (2006) J. Biol. Chem., 281:1580-1586), the data further suggest a dominant-negative effect of the variant TRPV4^P19S allele. These data indicate that this polymorphism in the TRPV4 gene affects channel function in vivo and influences systemic water balance on a population-wide basis in humans. Thus, the data provided herein indicates that the non-synonymous polymorphism as the cause for hyponatremia, potentially via reducing the hypothalamic osmolality set-point. The data provided herein also indicates a stronger association between the presence of the TRPV4^P19S allele and hyponatremia in male subjects. In an earlier study, male sex was associated with mild or moderate hyponatremia at presentation to hospital or during hospitalization (Hawkins, R. C. (2003) Clin. Chim. Acta., 337:169-72); however, women may be more susceptible to permanent brain damage in response to acute hyponatremia (Ayus et al. (2008) Am. J. Physiol. Renal. Physiol., 295: F619-24) . Sex hormones may influence TRPV4 function in vivo, as has been observed for TRPM6 in vitro (Cao et al. (2009) J. Biol. Chem., 284:14788-14795) .

A second nonsynonymous single nucleotide polymorphism (a G>C change (i.e., the GCG codon for alanine to CCG codon for proline) in the TRPV4 gene gives rise to an Ala-to-Pro substitution at residue 10. Association of this human polymorphism with serum sodium concentration and with hyponatremia is also demonstrated herein. The alteration of residue 10 may be in combination with the above described alteration at position 19.

Although mammalian osmoregulation is incompletely understood, abundant data point to a role for TRPV4 as a component of the central osmosensing mechanism. As stated hereinabove, TRPV4 was cloned on the basis of its homology with the C. elegans neural tonicity-sensing channel, OSM-9 (Liedtke et al. (2000) Cell, 103:525-35; Strotmann et al. (2000) Nat. Cell Biol., 2:695-702). TRPV4 is activated by hypotonicity in vitro - and perturbations of even a few mOsmol/kg H₂O were sufficient to achieve this effect (Liedtke et al. (2000) Cell, 103:525-35; Strotmann et al. (2000) Nat. Cell Biol., 2:695-702; Wissenbach et al. (2000) FEBS Lett., 485:127- 34) . Such exquisite sensitivity closely parallels the in vivo mechanism whereby a change of only a few mOsmol/kg H₂O influences release of arginine vasopressin. In rodents, TRPV4 is expressed in the bloodbrain barrier-deficient central osmosensing nuclei (Liedtke et al. (2000) Cell, 103:525-35; Guler et al. (2002) J. Neurosci., 22:6408-14), and targeted deletion of the TRPV4 gene gives rise to aberrant osmoregulation in murine models (Mizuno et al. (2003) Am. J. Physiol. Cell Physiol., 285 :C96-C101; Liedtke et al. (2003) Proc. Natl. Acad. Sci., 100:13698-703) . In addition, an as- of-yet unidentified splice variant of the closely related TRPVl channel likely represents the central sensor of hypertonicity (Naeini et al. (2006) Nat. Neurosci., 9:93-8) . In aggregate, these data strongly support a role for TRPV4 in the regulation of systemic water balance.

With respect to the TRPV4 gene, the loss-of- function alleles provided herein are specifically associated with hyponatremia. This phenotype is supported by observations made with in vitro hypotonicity responsiveness of both heterologously expressed (Liedtke et al. (2000) Cell, 103:525-35; Strotmann et al. (2000) Nat. Cell Biol., 2:695-702; Wissenbach et al. (2000) FEBS Lett., 485:127-34) and natively expressed (Chung et al. (2003) J. Biol. Chem., 278:32037-46; Wu et al. (2007) Am. J. Physiol. Renal Physiol., 293 : F1699-713) TRPV4 channels. Reduced sensitivity to systemic hypotonicity should lead to a partial failure of hypotonicity-correcting action (e.g., an inability to completely shut off arginine vasopressin release) . However, TRPV4-null mice exhibit a variable osmotic phenotype. Mizuno et al. noted no difference in plasma sodium concentration or in circulating levels of arginine vasopressin in TRPV4^~/" mice, relative to their wild-type littermates (Mizuno et al. (2003) Am. J. Physiol. Cell Physiol., 285 :C96-C101) . Provocative testing with water loading also failed to uncover a defect. Hyperosmotic challenge in this model - via simultaneous water restriction and intraperitoneal propylene glycol - resulted in an enhanced arginine vasopressin response (Mizuno et al. (2003) Am. J. Physiol. Cell Physiol., 285:C96-C101) . Liedtke et al. similarly noted no gross difference in plasma osmolality in TRPV4-null mice, compared to wild-type; however, when mice were single-housed, the TRPV4^"/" mice exhibited a 5 mOsmol/kg H₂O increment in plasma osmolality (Liedtke et al. (2003) Proc. Natl. Acad. Sci . , 100:13698-703) . Opposite the findings of the Mizuno group, these investigators noted an exaggerated arginine vasopressin response to osmotic challenge (albeit with a different stimulus - intraperitoneal hypertonic NaCl) (Liedtke et al. (2003) Proc. Natl. Acad. Sci., 100:13698-703) . Importantly, during chronic treatment with exogenous vasopressin analog, the TRPV4^"/" mice exhibited a much more robust drinking response and much more dramatic fall in blood osmolality; the net effect was hypotonicity in the TRPV4^"7' mice, relative to both their own baseline and that of their wild-type littermates (Liedtke et al. (2003) Proc. Natl. Acad. Sci., 100:13698-703) . In sum, they drink too little in the absence of unregulated vasopressin and too much in the presence of unregulated vasopressin. Therefore, hypofunctioning of the TRPV4 allele (s) predisposes to hyponatremia in the presence of constitutive vasopressin action. These data, coupled with the data provided herein, indicate that presence of the TRPV4^P19S allele synergizes with human conditions marked by chronically upregulated vasopressin level or vasopressin effect in promoting hyponatremia.

Notably, the human TRPV4^P19S allele is not a null allele; rather, it is hypofunctioning (abnormally low or inadequate functioning) and a subject homozygous for this allele has not yet been identified. In addition, in vitro function of the variant channel represented by the TRPV4^P19S allele in response to some agonists is fully preserved (Fig. 2) . Moreover, an element of TRPV4 osmoregulatory function may be exerted peripherally rather than centrally, through expression of the channel in the kidney collecting duct (Wu et al. (2007) Am. J. Physiol. Renal Physiol., 293: F1699-713; Tian et al. (2004) Am. J. Physiol. Renal Physiol., 287:F17-24) . Here, AQP2-dependent apical water permeability (i.e., water retention) is actively modulated by arginine vasopressin. Complete absence of TRPV4 from this nephron segment - pivotal to urinary concentration - may influence water balance independent of a central osmosensory function. Therefore, a TRPV4^"7" background is likely not functionally synonymous with the presence of a single hypofunctioning TRPV4^P19S allele in humans. The molecular mechanism through which the Pro-to- Ser substitution at residue 19 reduces osmoresponsiveness of the human TRPV4 channel is not completely clear. Because introduction of this serine gives rise to a high-probability context for protein phosphorylation (NetPhos prediction server; www.cbs.dtu.dk/services/NetPhos/; Blom et al. (1999) J. MoI. Biol., 294:1351-1362), this residue may undergo posttranslational modification only in the variant allele.

As described herein, hyponatremia was assigned based upon serum sodium concentration. No assessment had been made as to whether subjects were symptomatically hyponatremic at the time their laboratory studies were performed. Although the rate of change in serum sodium concentration may impact the development of symptoms (Adrogue et al. (2000) N. Engl. J. Med., 342:1581-9), even modest "stable" hyponatremia leads to impaired functioning of the central nervous system (Renneboog et al. (2006) Am. J. Med., 119:71 el- 8; Schrier et al. (2006) N. Engl. J. Med., 355:2099- 112) . Screening for the presence of the mutant alleles of the instant invention (e.g., TRPV4^P19S and TRPV4^A10P) is justified in human subjects as an index of propensity to aberrant water balance, irrespective of their present serum sodium concentration. The alleles may also synergize with environmental risk factors in the development of overt hyponatremia. Subjects may be predisposed to hyponatremia by virtue of their postoperative state, medication usage, or recreational activities (Verbalis et al. (2007) in Brenner and Rector's The Kidney, eds. Brenner BM, Rector

FC (Saunders Elsevier, Philadelphia), 8th Ed, pp 459- 504) . For example, some patients who are treated with psychotropic medications or diuretic agents develop a marked reduction in their plasma sodium level. This is a manifestation of excess body water relative to salt content. Other patients, when administered by vein or the gastrointestinal tract relatively large amounts of dilute fluids fail to adequately eliminate the excess water (Verbalis, J. G., Hyponatremia and hypoosmolar disorders, in Primer on Kidney Diseases, ed. Greenberg, A., Third ed., San Diego, Academic Press, 2001, pp 57- 63) . Some medications commonly used for treating hypertension and mental health disorders cause water retention and hyponatremia in a small subset of patients in an unpredictable fashion (Sonnenblick et al. (1993) Chest, 103:601-606; Liu et al. (1996) Cmaj , 155:519- 527) . Drugs/medications that have been shown to cause hyponatremia include, without limitation, acetazolamide, amiloride, amphotericin, aripiprazole, atovaquone, thiazide diuretics, amiodarone, basiliximab, angiotensin II receptor blockers, angiotensin-converting enzyme inhibitors, bromocriptine, carbamazepine, carboplatin, carvedilol, celecoxib, cyclophosphamide, clofibrate, desmopressin, donepezil, duloxetine, eplerenone, gabapentin, haloperidol, heparin, hydroxyurea, indapamide, indomethacin, ketorolac, levetiracetam, loop diuretics, lorcainide, mirtazapine, mitoxantrone, nimodipine, oxcarbazepine, opiates, oxytocin, pimozide, propafenone, proton pump inhibitors, quetiapine, sirolimus, ticlopidine, tolterodine, vincristine, selective serotonin reuptake inhibitors, sulfonylureas, trazodone, tolbutamide, venlafaxine, zalcitabine, and zonisamide . Similarly, some individuals who consume large amounts of hypotonic (dilute) fluids during heavy exercise (e.g., marathon road racing) retain too much water and develop low plasma sodium levels (Almond et al. (2005) N. Engl. J. Med., 352:1550-1556) . The elderly, in general, are predisposed to water retention (Ayus et al. (1996) Semin. Nephrol., 16:277-288) . Such water retention can cause abnormal thinking and behavior, seizures, coma, and death (Verbalis, J. G., Hyponatremia and hypoosmolar disorders, in Primer on Kidney Diseases, ed. Greenberg, A., Third ed., San Diego, Academic Press, 2001, pp 57-63) .

In patients lacking obvious medical conditions known to predispose to abnormal water retention (e.g., heart, kidney, or liver failure) , there is no known way to reliably identify which individuals are at increased risk for this potentially life-threatening complication. Accordingly, screening is particularly valuable in subjects predisposed to hyponatremia by virtue of their post-operative state, medication usage, or recreational activities (Verbalis et al., (2007) Disorders of Water Balance. In: Brenner BM, ed. Brenner and Rector's The Kidney: Saunders) . As an example, assessment of the SNP could be performed before patients, particularly the elderly, were treated with the commonly prescribed antihypertensive agent, hydrochlorothiazide, or before SSRI-type antidepressant medication was prescribed. Of note, the greater frequency of the TRPV4^P19S allele among the Yoruba of Nigeria (The International HapMap Project (2003) Nature, 426:789-96) and among African American subjects (present data) may reflect the selective advantage of a modest water excess (i.e., a lower setpoint for systemic osmolality) in conferring protection from symptomatic water deficit in hot environments in which water access may be unpredictable.

Hyponatremia may be treated through a variety of ways. Hypertonic saline (e.g., 3% saline) may be used to rapidly increase serum sodium level in patients with severe acute or chronic hyponatremia. Arginine vasopressin antagonists may also be used to treat hyponatremia through V₂ antagonism of AVP in the renal collecting ducts, thereby resulting in aquaresis (excretion of free water) . Conivaptan (Vaprisol®) is an arginine vasopressin antagonist (V_IA, V₂) indicated for euvolemic (dilutional) and hypervolemic hyponatremia. Those subjects identified as having the mutant alleles of the instant invention may be treated with the above methods, as required.

Definitions

The following definitions are provided to facilitate an understanding of the present invention:

"Pharmaceutically acceptable" indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

A "carrier" refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite) , solubilizer (e.g., Tween 80, Polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), water, aqueous solutions, oils, bulking substance (e.g., lactose, mannitol), excipient, auxilliary agent or vehicle with which an active agent of the present invention is administered. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, 20th Edition, (Lippincott, Williams and Wilkins) , 2000; Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

As used herein, the term "hyponatremia" refers to the condition wherein there is an excess of total body water relative to total body sodium content. In other words, hyponatremia may also refer to the condition of having an abnormally low extracellular concentration of sodium ions (the major cation of the extracellular fluid) . The extracellular concentration of sodium ions varies between individuals, but the average extracellular concentration of sodium is typically about 141-142 mEq/L. Hyponatremia is typically diagnosed when extracellular sodium concentration drops to 135 mEq/L or less. Thus, sodium concentration may be considered "abnormally low" when such concentration is at least 4% lower than the normal concentration, at least 5% lower, at least 6% lower, at least 7% lower, or more percent lower than the average extracellular concentration of sodium.

As used herein, a "biological sample" refers to a sample of biological material obtained from a subject, preferably a human subject, including a tissue, a tissue sample, a cell sample, a tumor sample, and a biological fluid (e.g., blood or urine) . A biological sample may be obtained in the form of, e.g., a tissue biopsy, such as, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy and an endoscopic biopsy. As used herein, "diagnose" refers to detecting and identifying a disease in a subject. The term may also encompass assessing or evaluating the disease status (progression, regression, stabilization, response to treatment, etc.) in a patient known to have the disease. As used herein, the term "prognosis" refers to providing information regarding the impact of the presence of a disease or disorder (e.g., as determined by the diagnostic methods of the present invention) on a subject's future health (e.g., expected morbidity or mortality, the likelihood of getting cancer, and the risk of metastasis) . In other words, the term "prognosis" refers to providing a prediction of the probable course and outcome of a disease/disorder or the likelihood of recovery from the disease/disorder. The term "treat" as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms) , delay in the progression of the condition, etc.

As used herein, a "conservative" amino acid substitution/mutation refers to substituting a particular amino acid with an amino acid having a side chain of similar nature (i.e., replacing one amino acid with another amino acid belonging to the same group) . A "non-conservative" amino acid substitution/mutation refers to replacing a particular amino acid with another amino acid having a side chain of different nature (i.e., replacing one amino acid with another amino acid belonging to a different group) . Groups of amino acids having a side chain of similar nature are known in the art and include, without limitation, basic amino acids (e.g., lysine, arginine, histidine) ; acidic amino acids (e.g., aspartic acid, glutamic acid); neutral amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) ; amino acids having a polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) ; amino acids having a non-polar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) ; amino acids having an aromatic side chain (e.g., phenylalanine, tryptophan, histidine); amino acids having a side chain containing a hydroxyl group (e.g., serine, threonine, tyrosine), and the like. The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains about 10-100, about 10-50, about 15-30, about 15-25, about 20-50, or more nucleotides, although it may contain fewer nucleotides. The probes herein may be selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target, although they may. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically. The term "primer" as used herein refers to an oligonucleotide, either RNA or DNA, either single- stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as appropriate temperature and pH, the primer may be extended at its 3^? terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically about 10-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3¹ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, non- complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.

Polymerase chain reaction (PCR) has been described in U.S. Patents 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which are incorporated by reference herein.

With respect to single stranded nucleic acids, particularly oligonucleotides, the term "specifically hybridizing" refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single- stranded nucleic acids of non-complementary sequence. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.

For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press) :

Tm = 81.5°C + lβ.βLog [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex

As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57⁰C. The Tm of a DNA duplex decreases by 1 - 1.5⁰C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42⁰C. The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25⁰C below the calculated Tm of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12 20⁰C below the Tm of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42⁰C, and washed in 2X SSC and 0.5% SDS at 55⁰C for 15 minutes. A high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt ' s solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42⁰C, and washed in IX SSC and 0.5% SDS at 65⁰C for 15 minutes. A very high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt ' s solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42°C, and washed in 0. IX SSC and 0.5% SDS at 65⁰C for 15 minutes. The term "isolated" may refer to a compound or complex that has been sufficiently separated from other compounds with which it would naturally be associated. "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with fundamental activity or ensuing assays, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition of the invention for performing a method of the invention. The phrase "solid support" refers to any solid surface including, without limitation, any chip (for example, silica-based, glass, or gold chip) , glass slide, membrane, plate, bead, solid particle (for example, agarose, sepharose, polystyrene or magnetic bead), column (or column material), test tube, or microtiter dish.

As used herein, the term "microarray" refers to an ordered arrangement of hybridizable array elements. The array elements are arranged so that there are at least one or more different array elements on a solid support. Preferably, the array elements comprise oligonucleotide probes .

An "antibody" or "antibody molecule" is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. The term includes polyclonal, monoclonal, chimeric, single domain (Dab) and bispecific antibodies. As used herein, antibody or antibody molecule contemplates recombinantly generated intact immunoglobulin molecules and immunologically active portions of an immunoglobulin molecule such as, without limitation: Fab, Fab', F(ab')2, F(v), scFv, scFv2, scFv-Fc, and the like.

TRPV4 Human TRPV4 is GenBank GenelD: 59341. Exemplary amino acid sequences of human TRPV4 are SEQ ID NOs: 1 and 2. A TRPV4 amino acid sequence may have 75%, 80%, 85%, 90%, 95%, 97%, or 99% homology with any of these amino acid molecules, particularly the longer form.

GenBank Accession No. EAW97878 (SEQ ID NQ: 1) MADSSEGPRA GPGEVAELPG DESGTPGGEA FPLSSLANLF EGEDGSLSPS PADASRPAGP GDGRPNLRMK FQGAFRKGVP NPIDLLESTL YESSVVPGPK KAPMDSLFDY GTYRHHSSDN KRWRKKIIEK QPQSPKAPAP QPPPILKVFN RPILFDIVSR GSTADLDGLL PFLLTHKKRL TDEEFREPST GKTCLPKALL NLSNGRNDTI PVLLDIAERT GNMREFINSP FRDIYYRGQT ALHIAIERRC

KHYVELLVAQ GADVHAQARG RFFQPKDEGG YFYFGELPLS LAACTNQPHI

VNYLTENPHK KADMRRQDSR GNTVLHALVA IADNTRENTK FVTKMYDLLL LKCARLFPDS NLEAVLNNDG LSPLMMAAKT GKIGNRHEML AVEPINELLR DKWRKFGAVS FYINVVSYLC AMVIFTLTAY YQPLEGTPPY PYRTTVDYLR LAGEVITLFT GVLFFFTNIK DLFMKKCPGV NSLFIDGSFQ LLYFIYSVLV IVSAALYLAG IEAYLAVMVF ALVLGWMNAL YFTRGLKLTG TYSIMIQKIL FKDLFRFLLV YLLFMIGYAS ALVSLLNPCA NMKVCNEDQT NCTVPTYPSC RDSETFSTFL LDLFKLTIGM GDLEMLSSTK YPVVFIILLV TYIILTFVLL LNMLIALMGE TVGQVSKESK HIWKLQWATT ILDIERSFPV FLRKAFRSGE MVTVGKSSDG TPDRRWCFRV DEVNWSHWNQ NLGIINEDPG KNETYQYYGF SHTVGRLRRD RWSSVVPRVV ELNKNSNPDE VVVPLDSMGN PRCDGHQQGY PRKWRTDDAP L

GenBank Accession No. EAW97879 (SEQ ID NO: 2) MADSSEGPRA GPGEVAELPG DESGTPGGEA FPLSSLANLF EGEDGSLSPS PADASRPAGP GDGRPNLRMK FQGAFRKGVP NPIDLLESTL YESSVVPGPK KAPMDSLFDY GTYRHHSSDN KRWRKKIIEK QPQSPKAPAP QPPPILKVFN RPILFDIVSR GSTADLDGLL PFLLTHKKRL TDEEFREPST GKTCLPKALL NLSNGRNDTI PVLLDIAERT GNMREFINSP FRDIYYRGQT ALHIAIERRC

KHYVELLVAQ GADVHAQARG RFFQPKDEGG YFYFGELPLS LAACTNQPHI

VNYLTENPHK KADMRRQDSR GNTVLHALVA IADNTRENTK FVTKMYDLLL LKCARLFPDS NLEAVLNNDG LSPLMMAAKT GKIGIFQHII RREVTDEDTR HLSRKFKDWA YGPVYSSLYD LSSLDTCGEE ASVLEILVYN SKIENRHEML AVEPINELLR DKWRKFGAVS FYINVVSYLC AMVIFTLTAY YQPLEGTPPY PYRTTVDYLR LAGEVITLFT GVLFFFTNIK DLFMKKCPGV NSLFIDGSFQ LLYFIYSVLV IVSAALYLAG IEAYLAVMVF ALVLGWMNAL YFTRGLKLTG TYSIMIQKIL FKDLFRFLLV YLLFMIGYAS ALVSLLNPCA NMKVCNEDQT NCTVPTYPSC RDSETFSTFL LDLFKLTIGM GDLEMLSSTK YPVVFIILLV TYIILTFVLL LNMLIALMGE TVGQVSKESK HIWKLQWATT ILDIERSFPV FLRKAFRSGE MVTVGKSSDG TPDRRWCFRV DEVNWSHWNQ NLGIINEDPG KNETYQYYGF SHTVGRLRRD RWSSVVPRVV ELNKNSNPDE VVVPLDSMGN PRCDGHQQGY PRKWRTDDAP L

GenBank Accession No. NM 147204 (SEQ ID NO: 3) atggcggattc cagcgaaggc ccccgcgcgg ggcccgggga ggtggctgag ctccccgggg atgagagtgg caccccaggt ggggaggctt ttcctctctc ctccctggcc aatctgtttg agggggagga tggctccctt tcgccctcac cggctgatgc cagtcgccct gctggcccag gcgatgggcg accaaatctg cgcatgaagt tccagggcgc cttccgcaag ggggtgccca accccatcga tctgctggag tccaccctat atgagtcctc ggtggtgcct gggcccaaga aagcacccat ggactcactg tttgactacg gcacctatcg tcaccactcc agtgacaaca agaggtggag gaagaagatc atagagaagc agccgcagag ccccaaagcc cctgcccctc agccgccccc catcctcaaa gtcttcaacc ggcctatcct ctttgacatc gtgtcccggg gctccactgc tgacctggac gggctgctcc cattcttgct gacccacaag aaacgcctaa ctgatgagga gtttcgagag ccatctacgg ggaagacctg cctgcccaag gccttgctga acctgagcaa tggccgcaac gacaccatcc ctgtgctgct ggacatcgcg gagcgcaccg gcaacatgcg ggagttcatt aactcgccct tccgtgacat ctactatcga ggtcagacag ccctgcacat cgccattgag cgtcgctgca aacactacgt ggaacttctc gtggcccagg gagctgatgt ccacgcccag gcccgtgggc gcttcttcca gcccaaggat gaggggggct acttctactt tggggagctg cccctgtcgc tggctgcctg caccaaccag ccccacattg tcaactacct gacggagaac ccccacaaga aggcggacat gcggcgccag gactcgcgag gcaacacagt gctgcatgcg ctggtggcca ttgctgacaa cacccgtgag aacaccaagt ttgttaccaa gatgtacgac ctgctgctgc tcaagtgtgc ccgcctcttc cccgacagca acctggaggc cgtgctcaac aacgacggcc tctcgcccct catgatggct gccaagacgg gcaagattgg gaaccgccac gagatgctgg ctgtggagcc catcaatgaa ctgctgcggg acaagtggcg caagttcggg gccgtctcct tctacatcaa cgtggtctcc tacctgtgtg ccatggtcat cttcactctc accgcctact accagccgct ggagggcaca ccgccgtacc cttaccgcac cacggtggac tacctgcggc tggctggcga ggtcattacg ctcttcactg gggtcctgtt cttcttcacc aacatcaaag acttgttcat gaagaaatgc cctggagtga attctctctt cattgatggc tccttccagc tgctctactt catctactct gtcctggtga tcgtctcagc agccctctac ctggcaggga tcgaggccta cctggccgtg atggtctttg ccctggtcct gggctggatg aatgcccttt acttcacccg tgggctgaag ctgacgggga cctatagcat catgatccag aagattctct tcaaggacct tttccgattc ctgctcgtct acttgctctt catgatcggc tacgcttcag ccctggtctc cctcctgaac ccgtgtgcca acatgaaggt gtgcaatgag gaccagacca actgcacagt gcccacttac ccctcgtgcc gtgacagcga gaccttcagc accttcctcc tggacctgtt taagctgacc atcggcatgg gcgacctgga gatgctgagc agcaccaagt accccgtggt cttcatcatc ctgctggtga cctacatcat cctcaccttt gtgctgctcc tcaacatgct cattgccctc atgggcgaga cagtgggcca ggtctccaag gagagcaagc acatctggaa gctgcagtgg gccaccacca tcctggacat tgagcgctcc ttccccgtat tcctgaggaa ggccttccgc tctggggaga tggtcaccgt gggcaagagc tcggacggca ctcctgaccg caggtggtgc ttcagggtgg atgaggtgaa ctggtctcac tggaaccaga acttgggcat catcaacgag gacccgggca agaatgagac ctaccagtat tatggcttct cgcataccgt gggccgcctc cgcagggatc gctggtcctc ggtggtaccc cgcgtggtgg aactgaacaa gaactcgaac ccggacgagg tggtggtgcc tctggacagc atggggaacc cccgctgcga tggccaccag cagggttacc cccgcaagtg gaggactgat gacgccccgc tctag

GenBank Accession No. NM 021625 (SEQ ID NO: 4) atggcggattc cagcgaaggc ccccgcgcgg ggcccgggga ggtggctgag ctccccgggg atgagagtgg caccccaggt ggggaggctt ttcctctctc ctccctggcc aatctgtttg agggggagga tggctccctt tcgccctcac cggctgatgc cagtcgccct gctggcccag gcgatgggcg accaaatctg cgcatgaagt tccagggcgc cttccgcaag ggggtgccca accccatcga tctgctggag tccaccctat atgagtcctc ggtggtgcct gggcccaaga aagcacccat ggactcactg tttgactacg gcacctatcg tcaccactcc agtgacaaca agaggtggag gaagaagatc atagagaagc agccgcagag ccccaaagcc cctgcccctc agccgccccc catcctcaaa gtcttcaacc ggcctatcct ctttgacatc gtgtcccggg gctccactgc tgacctggac gggctgctcc cattcttgct gacccacaag aaacgcctaa ctgatgagga gtttcgagag ccatctacgg ggaagacctg cctgcccaag gccttgctga acctgagcaa tggccgcaac gacaccatcc ctgtgctgct ggacatcgcg gagcgcaccg gcaacatgcg ggagttcatt aactcgccct tccgtgacat ctactatcga ggtcagacag ccctgcacat cgccattgag cgtcgctgca aacactacgt ggaacttctc gtggcccagg gagctgatgt ccacgcccag gcccgtgggc gcttcttcca gcccaaggat gaggggggct acttctactt tggggagctg cccctgtcgc tggctgcctg caccaaccag ccccacattg tcaactacct gacggagaac ccccacaaga aggcggacat gcggcgccag gactcgcgag gcaacacagt gctgcatgcg ctggtggcca ttgctgacaa cacccgtgag aacaccaagt ttgttaccaa gatgtacgac ctgctgctgc tcaagtgtgc ccgcctcttc cccgacagca acctggaggc cgtgctcaac aacgacggcc tctcgcccct catgatggct gccaagacgg gcaagattgg gatctttcag cacatcatcc ggcgggaggt gacggatgag gacacacggc acctgtcccg caagttcaag gactgggcct atgggccagt gtattcctcg ctttatgacc tctcctccct ggacacgtgt ggggaagagg cctccgtgct ggagatcctg gtgtacaaca gcaagattga gaaccgccac gagatgctgg ctgtggagcc catcaatgaa ctgctgcggg acaagtggcg caagttcggg gccgtctcct tctacatcaa cgtggtctcc tacctgtgtg ccatggtcat cttcactctc accgcctact accagccgct ggagggcaca ccgccgtacc cttaccgcac cacggtggac tacctgcggc tggctggcga ggtcattacg ctcttcactg gggtcctgtt cttcttcacc aacatcaaag acttgttcat gaagaaatgc cctggagtga attctctctt cattgatggc tccttccagc tgctctactt catctactct gtcctggtga tcgtctcagc agccctctac ctggcaggga tcgaggccta cctggccgtg atggtctttg ccctggtcct gggctggatg aatgcccttt acttcacccg tgggctgaag ctgacgggga cctatagcat catgatccag aagattctct tcaaggacct tttccgattc ctgctcgtct acttgctctt catgatcggc tacgcttcag ccctggtctc cctcctgaac ccgtgtgcca acatgaaggt gtgcaatgag gaccagacca actgcacagt gcccacttac ccctcgtgcc gtgacagcga gaccttcagc accttcctcc tggacctgtt taagctgacc atcggcatgg gcgacctgga gatgctgagc agcaccaagt accccgtggt cttcatcatc ctgctggtga cctacatcat cctcaccttt gtgctgctcc tcaacatgct cattgccctc atgggcgaga cagtgggcca ggtctccaag gagagcaagc acatctggaa gctgcagtgg gccaccacca tcctggacat tgagcgctcc ttccccgtat tcctgaggaa ggccttccgc tctggggaga tggtcaccgt gggcaagagc tcggacggca ctcctgaccg caggtggtgc ttcagggtgg atgaggtgaa ctggtctcac tggaaccaga acttgggcat catcaacgag gacccgggca agaatgagac ctaccagtat tatggcttct cgcataccgt gggccgcctc cgcagggatc gctggtcctc ggtggtaccc cgcgtggtgg aactgaacaa gaactcgaac ccggacgagg tggtggtgcc tctggacagc atggggaacc cccgctgcga tggccaccag cagggttacc cccgcaagtg gaggactgat gacgccccgc tctag

In accordance with the instant invention, primers and probes which specifically recognize at least one of the mutants of the instant invention (e.g., TRPV4^P19S and/or TRPV4^A10p) are also provided. The probes or primers may be used to detect the presence of and/or expression of at least one of the mutant alleles of the instant invention (e.g., TRPV4^P19S and/or TRPV4^A1QP) . In a particular embodiment, the nucleic acid probes or primers encompass a nucleic acid sequence encoding at least one of the P19S and AlOP mutations. In yet another embodiment, the probes and primers specifically hybridize to at least one of the mutant alleles of the instant invention (e.g., TRPV4^pl9s and/or TRPV4^Al0p) , optionally to the exclusion of wild-type TRPV4. Methods in which nucleic acid probes and primers may be utilized to identify the alleles of the instant invention include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR) (e.g., allele specific PCR and RT-PCR) . Other methods of detecting the mutant alleles of the instant invention include mutational analysis/conformation sensitive gel electrophoresis (CSGE) , linkage analysis (Thompson & Thompson, Genetics in Medicine, 5th ed, 1991, W. B. Saunders Company, Philadelphia; Lathrop, Proc. Nat. Acad. Sci. (1984) 81:3443-3446; T. Strachan, Chapter 4, "Mapping the human genome" in The Human Genome, 1992 BIOS Scientific Publishers Ltd. Oxford), allele specific oligonucleotide screening methods (Saiki et al., Nature (1986) 324:163-166), ligase mediated allele detection method (Landegren et al., Science (1988) 241:1077-1080), denaturing gradient gel electrophoresis (Chapter 7 of Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, W. H. Freeman and Co, New York (1992)), temperature gradient gel electrophoresis, single-strand conformation polymorphism analysis (Orita et al., Proc. Nat. Acad. Sci. (1989) 86:2766-2770), chemical or enzymatic cleavage of mismatches (Grompe et al., Am. J. Hum. Genet. (1991) 48:212-222), and non-PCR based DNA diagnostics.

In a particular embodiment for screening for the mutant alleles of the instant invention, the TRPV4 containing nucleic acid or a fragment thereof (e.g., one or more exons (e.g., exon 2)) in the sample may be initially amplified, e.g. using PCR, to increase the amount of the template as compared to other sequences present in the sample. This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample. The presence of a mutation may then be detected by sequencing (e.g., pyrosequencing) or use of a mutant allele probe or primer.

In addition to detecting hypofunctioning forms of TRPV4, such as TRPV4^P19S, by assaying nucleic acids encoding TRPV4, the TRPV4 protein may also be directly detected and/or isolated and sequenced. In a particular embodiment, mutant TRPV4 proteins (e.g. TRPV4^P19S and/or TRPV4^A1OP) may be detected with an antibody which is immunologically specific for the mutant TRPV4, optionally to the exclusion of wild-type TRPV4.

Further, assays for detecting a mutant TRPV4 may be conducted on any type of biological sample, including but not limited to body fluids (including blood, saliva, urine, serum) , any type of cell (such as blood cells) , or body tissue. In a particular embodiment, the biological sample is obtained by a non-invasive method such as by a buccal swab.

The probes, primers, and/or antibodies of the instant invention may be incorporated into a kit. The probes (e.g., TRPV4^P19S and/or TRPV4^A10P) may be immobilized on a solid support (e.g., a chip or microarray) . The kit may further comprise instruction material, buffers, and/or containers.

Cells and transgenic animals comprising a nucleic acid encoding at least one mutant allele of the instant invention (e.g., TRPV4^P19S and/or TRPV4^Λ1OP) are also encompassed by the instant invention. The term "transgenic animal" is intended to include any non-human animal, preferably vertebrate, in which one or more of the cells of the animal contain heterologous/exogenous nucleic acid encoding at least one mutant allele of the instant invention (e.g., TRPV4^P19S and/or TRPV4^Al0p) , optionally from a different species (e.g., human) . Non- human animals include without limitation, rodents, mice, rats, non-human primates, sheep, dog, cow, amphibians, zebrafish, reptiles, and the like. In a preferred embodiment, the animal is a mouse. In another embodiment, the transgenic animal comprising a heterologous nucleic acid encoding at least one mutant allele of the instant invention (e.g., TRPV4^P19S and/or TRPV4^Al0p) is hyponatremic compared to a wild-type mouse or at greater risk for hyponatremia compared to a wild- type mouse. Modifications and/or deletions may render the naturally occurring TRPV4 gene nonfunctional, thereby producing a "knock out" transgenic animal (e.g., TRPV4^"7") . The transgenic animal of the instant invention may comprise homozygous or heterozygous null mutations in the endogenous TRPV4 gene with the heterologous/exogenous nucleic acid encoding TRPV4^P19S. The transgenic animal of the instant invention may also be generated by homologous recombination wherein at least one of the wild-type alleles is replaced with at least one mutant allele of the instant invention (e.g., TRPV4^P19S and/or TRPV4^A10p) . The transgenic animal of the instant invention may comprise a genome comprising a disruption of the endogenous TRPV4 gene, wherein the TRPV4 gene disruption leads to the failure to express TRPV4^WT and/or a functional TRPV4^WT. The disruption of the endogenous TRPV4 can be obtained through the disruption (e.g., insertion of a nucleic acid sequence (e.g., selectable marker) or deletion (e.g., all or part) ) of at least one exon.

Transgenic animals of the instant invention may be useful for the establishment of a nonhuman model for diseases involving improper expression and/or regulation of TRPV4, e.g., hyponatremia. The transgenic animals may also be useful as in vivo models for drug screening studies for certain human diseases, and for eventual treatment of disorders or diseases associated with TRPV4.

The following examples provide illustrative methods of practicing the instant invention, and are not intended to limit the scope of the invention in any way.

EXAMPLE 1 METHODS

Genotyping - Healthy Aging cohort Banked genomic DNA was obtained from the Healthy Aging cohort of the National Institutes on Aging-funded Layton Aging and Alzheimer's Disease Center database at Oregon Health & Science University (Howieson et al. (1993) Neurology, 43:1882-6) . Individuals in this Healthy Aging cohort represented non-demented control subjects for longitudinal studies of the determinants of Alzheimer's Disease and other dementing conditions in the elderly (Howieson et al. (1993) Neurology, 43:1882- 6) . Genomic DNA was subjected to phi29-based whole- genome amplification (REPLI-g kit; QIAGEN, Valencia, CA) . The TRPV4 exon of interest was PCR-amplified using primers bracketing the TRPV4^P19S polymorphism (rs3742030) ; the amplicon was then subjected to sequencing with one of the original amplification primers in an automated sequencing platform (Applied Biosystems (Foster City, CA) ; Vollum Institute for Advanced Biomedical Research) . Presence of the TRPV4^P19S allele was detected by inspection of electropherograms using FinchTV software (Geospiza, Seattle, WA) . Genomic DNA from a total of 299 subjects was genotyped for the presence of the TRPV4^P19S allele. For eight subjects, genotyping was not successful (i.e., insufficient sample), leaving 291 successful genotypes (219 Caucasian and 72 African American subjects) .

Genotyping - Osteoporotic Fractures in Men (MrOS) Study

The Osteoporotic Fractures in Men (MrOS) Study was designed to assess the determinants of fracture in 5995 healthy community-dwelling U.S. male subjects over 65 years of age (Orwoll et al. (2005) Contemp. Clin. Trials, 26:569-85) . Subjects were recruited from six centers (see Table 3; Blank et al. (2005) Contemp. Clin. Trials, 26:557-68) . Banked serum and genomic DNA were obtained by the parent study from 5532 subjects; all were male. Serum sodium, creatinine, and glucose were measured in all subjects on a single instrument using thawed previously-frozen serum (Clinical Laboratory, Portland V. A. Medical Center) . Subjects with serum creatinine > 1.3 were excluded from further analysis because abnormal renal function may lead to impaired water excretion (e.g., Yee et al. (1999) Chest, 115-.149S-157S) . Subjects with serum glucose ≥ 150 mg/dl were excluded because the independent osmotic effect of hyperglycemia depresses serum sodium concentration, rendering the measurement less reliable (e.g., Katz, M. A. (1973) N. Engl. J. Med., 289:843-4) . Genomic DNA from 1524 subjects was requested from the parent study, and 1449 samples were received. These represented subjects in one of three groups, based upon serum sodium concentration (see Fig. ID) . The "low" sodium concentration group was designed to include all subjects with serum sodium concentration ≤ 138 mEq/1, and the "high" sodium concentration group included all subjects with sodium ≥ 145 mEq/1. These groups approximated the lowest and highest deciles (or ~ 1.5 SD units) of the MrOS population, in terms of serum sodium concentration. The population mean for serum sodium concentration in non-excluded non-Hispanic Caucasian MrOS subjects (Table 3) was 141.4 mEq/1; for the "mean" group, every third subject was genotyped when subjects with serum sodium concentration of 141 and 142 mEq/1 were ordered by serum sodium concentration, and then by coded alphanumeric identifier (sodium concentrations were "binned" as integers at the time of reporting by the clinical laboratory) . Banked genomic DNA was subjected to phi29- based whole-genome amplification and genotyped for the presence of the TRPV4^P19S allele in a blinded fashion using a custom-designed real-time PCR-based assay directed against SNP rs3742030 (Applied Biosystems) . Of 1449 samples obtained from the parent study, 26 samples could not be genotyped (i.e., insufficient quantity of DNA) . The successfully genotyped non-Hispanic Caucasian subjects (n = 1304) were used for replication.

For both Healthy Aging and MrOS, all genotyping studies using human DNA were approved by the Institutional Review Board of the Portland V. A. Medical Center, or were deemed exempt by this body under Code of Federal Regulations, Title 45 - Public Welfare, Department of Health and Human Services; Part 46 - Protection of Human Subjects; Paragraph 46.101 (b) (4) - i.e. , Exemption 4. At the outset of this study, two non-synonymous polymorphisms in the human TRPV4 gene were listed in the public domain. One, rsllO68298, coded for a variant allele of TRPV4 with an Ala-to-Thr substitution at amino acid residue 565; however, no data on minor allele frequency were available. The second, rs3742030, coded for a variant allele with a Pro-to-Ser substitution at residue 19. A panel of anonymized genomic DNA samples of various ethnicities was screened and the rsllO68298 minor allele was not detected in any subjects. In contrast, the rs3742030 minor allele was detected in -5% of these samples, consistent with data reported in the International HapMap Project (The International HapMap Project (2003) Nature, 426:789-96) . Therefore, this polymorphism was focused on in all subsequent studies.

Statistical analysis

Unadjusted association between one copy of the variant allele and the presence of hyponatremia was determined via Chi-squared contingency table analysis with the Fisher Exact probability test. In the case of the MrOS cohort, the Yates correction for small cell number was applied because of the large size of the population. Comparison between mean serum sodium concentration in the presence and absence of the TRPV4^P19S allele was performed via two-tailed t-test for the Healthy Aging populations (non-Hispanic Caucasian and African American) , and via one-tailed t-test for the confirmatory MrOS population. Hyponatremia was defined as serum sodium concentration ≤ 135 mEq/1. Prevalence ratios were calculated in SAS using all covariates (see below) using a binomial distribution with a log link function for the MrOS cohort, and a Poisson distribution with a log link function for the Healthy Aging cohort (where there was a lack of converge with the Poisson distribution) .

For linear regression analysis to test the association between serum sodium concentration as a continuous variable and rs3742030 genotype in the non- Hispanic Caucasian Healthy Aging cohort, the data set was filtered to eliminate genotyped subjects with glucose ≥ 150 mg/dl (n = 11) to preserve consistency with the tested MrOS samples (where subjects with serum glucose ≥ 150 mg/dl were excluded) . In addition, two subjects were missing data on age; therefore, the final numbers for analysis were 76 males and 130 females. Covariates for linear regression included sex, age, and serum glucose concentration/ the latter two may impact serum sodium concentration (Katz, M. A. (1973) N. Engl. J. Med., 289:843-4; Miller, M. (2006) J. Am. Geriatr. Soc, 54:345-53) . Note that there was no serum creatinine determination for this data set. For female subjects, age and glucose concentration were significantly associated with serum sodium concentration (p < 0.0001 and p = 0.006, respectively); for male subjects, age was associated with serum sodium concentration (p = 0.0045) .

In the non-Hispanic Caucasian subset of the MrOS cohort, the unadjusted association between hyponatremia (sodium ≤ 135 mEq/1) and the "low", "mean", and "high" sodium groups did not reach statistical significance (p = 0.22, Pearson's Chi-squared test) . For linear regression analysis in this cohort, no subjects with serum glucose ≥ 150 mg/dl were genotyped and hence none required exclusion from the final analysis. Covariates for linear regression included age, serum glucose concentration, serum creatinine concentration, and recruitment center. Age and creatinine level were significantly associated with hyponatremia (p-value = 0.026 and < 0.0001, respectively) . One recruitment center (BI, Birmingham) was associated with hyponatremia (p = 0.022) whereas glucose concentration and other recruitment centers were not significantly associated with hyponatremia.

No adjustment was made for multiple comparisons because rs3742030 was the only polymorphism genotyped in these populations. Of note, no subjects homozygous for the TRPV4^P19S allele were identified in any study population, although only approximately three would be expected by Hardy-Weinberg equilibrium using the allelic frequency in the largest population (MrOS) .

Cell transfection Human TRPV4 cDNA was amplified from human kidney mRNA, cloned (with its native stop codon intact) into the mammalian expression vector pcDNA3. l/V5-His-TOPO, and confirmed by complete sequencing. The TRPV4^P19S polymorphism was introduced via site-directed mutagenesis (QuikChange®; Stratagene, La Jolla, CA) and the entire cDNA was confirmed by sequencing.

HEK293 cells were transiently transfected with polyethylenimine ExGen500 (Fermentas MBI, Burlington, Ontario, Canada) using 8 equivalents of polyethylemine together with 0.3 μg of pEGFPNl, and 3 μg of pcDNA3-

TRPV4WT or mutant TRPV4^P19S. Cells were used 12-48 hours after transfection.

Confocal immunofluorescence detection of TRPV4 was carried out as previously described (Arniges et al. (2006) J. Biol. Chem., 281:1580-1586; Lorenzo et al. (2008) Proc. Natl. Acad. Sci . USA, 105:12611-12616) .

Electrophysiological recordings Cationic currents were registered using the patch- clamp technique in whole-cell configuration. Patch pipettes were filled with a solution containing (in mM) : 20 CsCl, 100 Cs-acetate, 1 MgCl₂, 0.1 EGTA, 10 HEPES, 4 Na₂ATP and 0.1 NaGTP; 300 mosmoles/liter, pH 7.2-7.3. Pipette solutions containing 156 nM EET (Biomol;

Plymouth Meeting, PA) were also used where indicated. Cells were bathed in isotonic solutions containing (in mM) : 100 NaCl, 6 CsCl, 1 MgCl₂, 1 EGTA, 5 glucose, 10 HEPES and SO D-mannitol (315 mosmoles/1, pH 7.35) . The 15% (270 mosmoles/liter) and 30% hypotonic (220 mosmoles/liter) bathing solutions were prepared by reducing D-mannitol from the isotonic solution- Currents in response to 4aPDD and EET were obtained using isotonic bathing solutions containing (in mM) : 140 NaCl, 5 KCl, 1 MgC12, 10 HEPES and 1 EGTA (-310 mosmoles/liter, pH 7.3-7.4) . HEK293 were clamped at 0 mV and ramps from -100 mV to +100 mV (400 ms) were applied at a frequency of 0.2 Hz. Ramp data were acquired at 10 KHz and low-pass filtered at 1 KHz. All experiments were carried out at room temperature. Only those cells that presented GFP fluorescence were recorded. All experiments were carried out at room temperature.

Cell surface biotinylation experiments were performed as previously described (Xu et al. (2006) Am. J. Physiol., 290:F1103-F1109) . HEK293 cells were transiently transfected with full-length cDNA coding for wild-type human TRPV4 (hTRPV4) or for human TRPV4 point- mutated to incorporate either the A565T or the P19S nonsynonymous polymorphism. Whole-cell detergent lysates were resolved via SDS-PAGE and subjected to anti-TRPV4 immunoblotting as previously described (Xu et al. (2003) J. Biol. Chem. , 278:11520-11527) . Cells transfected in parallel were also subjected to cell- surface biotinylation and avidin-affinity precipitation, followed by anti-TRPV4 immunoblotting as previously described (Xu et al. (2006) Am. J. Physiol., 290 : F1103-F1109) .

RESULTS

An association between serum sodium concentration and a non-synonymous single nucleotide polymorphism (SNP) in the TRPV4 gene was tested. This polymorphism, rs3742030, gives rise to a non-conservative amino acid substitution (i.e., Pro-to-Ser) at residue 19; it was the only TRPV4 non-synonymous SNP for which minor allele frequency was reported in the International HapMap data set at the time these studies commenced (The International HapMap Project (2003) Nature, 426:789-96) . Banked genomic DNA from a panel of healthy elderly subjects (the Healthy Aging cohort) in the National Institutes on Aging-funded Layton Aging and Alzheimer's Disease Center database at Oregon Health & Science University (Howieson et al. (1993) Neurology, 43:1882-6) was genotyped for the presence of this allele (see Methods) . Characteristics of this cohort are presented in Table 1. Among non-Hispanic Caucasian subjects in this cohort (n = 219 successful genotypes) , the TRPV4^P19S allele appeared to be over-represented in subjects with the lowest serum sodium concentrations (Fig. IA) . A similar phenomenon was observed in the African American subjects from this cohort (Fig. IB/ n = 72) . Of note, the prevalence of the heterozygous state for this allele in Caucasian subjects (i.e., Utah residents with ancestry from northern and western Europe; CEU) and in the Yoruba in Ibadan, Nigeria (YRI) in the International HapMap Project data set (The International HapMap Project (2003) Nature, 426:789-96) is 0.017 and 0.100, respectively.

Table 1: Characteristics of the Healthy Aging cohort and of the Caucasian and African American subjects who were successfully genotyped. Characteristics of the Healthy Aging cohort and of the successfully genotyped non- Hispanic Caucasian and African American subjects, including number of subjects per group (n) , self- reported ethnicity, percent of subjects that were male, age (mean ± SD) , and laboratory values (mean ± SD) . Not all subjects were genotyped, owing to availability of genomic DNA. Subgroups include: i) all successfully genotyped non-Hispanic Caucasian subjects; and ii) all successfully genotyped African American subjects. Where indicated (*), two subjects were excluded from these calculations because a numerical value for age was not reported. Serum creatinine was not determined in this cohort. Prevalence of hyponatremia (serum sodium concentration ≤ 135 mEq/1) by genotype and ethnicity is shown in Fig. IE. Hyponatremia was associated with the TRPV4^P19S allele (p = 0.05 and 0.01 for the non-Hispanic Caucasian and African American populations, respectively, via Pearson's Chi-squared analysis) . For each cohort, the mean serum sodium concentration was significantly lower in the TRPV4^pl9s-positive subjects (by 1.6 and 2.4 mEq/1; p = 0.05 and 0.014 for the non- Hispanic Caucasian and African American populations, respectively, by t-Test; Fig. IF) . The effect of this allele upon systemic water balance was further quantified in the larger non-Hispanic Caucasian population using the covariates of age, sex, and serum glucose concentration; glucose exerts an osmotic effect independent of serum sodium concentration (Katz, M. A. (1973) N. Engl. J. Med., 289:843-4) and age may be associated with hyponatremia (Miller, M. (2006) J. Am. Geriatr. Soc, 54:345-53). The strength of association between serum sodium concentration (as a continuous variable) and rs3742030 genotype was determined by linear regression using the above covariates. Presence of the rs3742030 minor allele (i.e., the TRPV4^P19S allele) was significantly associated with serum sodium concentration for males (p = 0.0024) but less for females (p-value = 0.40) . Prevalence ratio calculations indicated that male subjects with the minor allele were 6.45 times as likely to exhibit hyponatremia as male subjects without the minor allele (95% CI: 1.22 - 34.25; p-value = 0.029), after inclusion of the covariates; for female subjects, the prevalence ratio was 1.76 (95% CI: 0.52 - 6.0; p-value = 0.37; Table 2) .

Table 2: Strength of association of serum sodium concentration with presence of the TRPV4^P19S allele in non-Hispanic Caucasian subjects genotyped in the Healthy Aging and MrOS cohorts, as tested via linear regression analysis on available covariates (shown) and stratified by sex (see Methods) . For the Healthy Aging cohort, subjects with missing age data (n = 2) were excluded from this analysis; in addition, subjects with serum glucose ≥150 mg/dL (n = 11) were excluded to maintain consistency with inclusion criteria for the genotyped MrOS cohort. Prevalence ratios (and 95% confidence intervals) were calculated for the presence of hyponatremia (serum sodium concentration ≤135 mEq/L) as a function of the presence of the TRPV4P19S allele, and incorporating available covariates. Male subjects with the TRPV4P19S allele in the Healthy Aging and MrOS cohorts were 6.45 and 2.43 times as likely, respectively, to exhibit hyponatremia as were subjects lacking the allele.

A larger male population was sought for replication of these findings. Banked genomic DNA was obtained from subjects enrolled in the Osteoporotic Fractures in Men Study (MrOS; see Methods), a prospective U.S. cohort study of 5995 community dwelling men aged 65 years and over (Orwoll et al. (2005) Contemp. Clin. Trials, 26:569-85) . Subjects with abnormal kidney function (i.e., serum creatinine > 1.3 mg/dl) and serum glucose > 150 mg/dl were excluded because renal insufficiency and marked hyperglycemia independently impact serum sodium concentration (Yee et al. (1999) Chest, 115 : 149S-157S; Katz, M. A. (1973) N. Engl. J. Med., 289:843-4) . In addition, the majority of participants in MrOS were non- Hispanic Caucasian; only subjects of this self-reported ethnicity were selected for the replication study. Characteristics of these subjects (n = 4305) are shown in Table 3. Serum sodium concentration followed a roughly normal distribution (Fig. ID) . All subjects with the lowest serum sodium concentration (i.e., ≤ 138 itiEq/1, corresponding to the lowest decile, or ~1.5 SD units below the population mean) and highest serum sodium concentration (i.e., ≥ 145 mEq/1, approximating the highest decile, or ~ 1.5 SD units above the population mean) were genotyped for the TRPV4^P19S allele, as was as a random selection of subjects with sodium concentration approximating the sample mean (141 - 142 mEq/1; see Methods) . This approach was adopted to ensure maximal representation of the population extremes, vis-a-vis serum sodium concentration. Characteristics of these subgroups are shown in Table 3. Prevalence of the TRPV4^P19S allele expressed as a function of serum sodium concentration is shown in Fig. 1C; prevalence of the TRPV4^P19S allele in the "low," "mean," and "high" serum sodium concentration groups in this cohort was 6.1% (n = 444), 4.2% (n= 448), and 3.7% (n = 408), respectively (Fig. ID, inset) . Mean serum sodium concentration was 0.9 mEq/1 lower in subjects with the TRPV4^P19S allele (Fig. IF; p = 0.04 via t-Test) . Serum sodium concentration was again significantly associated with the TRPV4^P19S allele (p = 0.019), as determined by linear regression analysis using the covariates of age, serum glucose, serum creatinine, and recruitment center (see Table 3 and Methods) . Subjects with the minor allele were 2.43 times as likely to exhibit hyponatremia as subjects without the allele (95% CI: 1.17 - 5.06; p- value = 0.017), after inclusion of the covariates in this exclusively male population (Table 2) .

Table 3: Characteristics of the MrOS cohort and of the serum sodium concentration subgroups who were successfully genotyped. Characteristics of the MrOS cohort and of the MrOS Low, Mean, and High serum sodium concentration subgroups, including number of subjects per group (n) , percent of subjects that were male, age (mean ± SD) , self-reported ethnicity, and laboratory values (mean ± SD) . The last six columns indicate percent of subjects from each MrOS recruitment site, where BI, MN, PA, PI, PO, and SD represent the MrOS Birmingham, Minneapolis, Palo Alto, Pittsburgh, Portland, and San Diego recruitment sites, respectively. Subgroups include all successfully genotyped non- Hispanic Caucasian subjects; they were drawn from the "All MrOS" pool based upon serum sodium concentration, as explained in Methods. "All MrOS" includes all subjects from the original MrOS cohort (n = 5995) who fulfilled the following criteria: i) serum sodium, creatinine, and glucose concentrations were determined; ii) ethnicity was self-reported as non-Hispanic Caucasian; iii) serum creatinine was ≤ 1.3 mg/dl; and iv) serum glucose was < 150 mg/dl.

It was sought to establish that the aberrant water balance associated with the TRPV4^P19S allele was not attributable to another polymorphism in tight linkage disequilibrium. All TRPV4 exons and exon-intron boundaries were re-sequenced from ten hyponatremic subjects with the TRPV4P19S minor allele; no other synonymous or otherwise functional polymorphisms were detected. In addition, no polymorphisms in strong linkage disequilibrium with rs3742030 impacting coding or splicing were identified in haplotype analysis (www.hapmap.org; International HapMap Project; Release 21) . Of note, no subjects with two copies of the rs3742030 minor (TRPV4^pl9s) allele were identified in the healthy Aging or MrOS populations.

In aggregate, these data indicated that the TRPV4^P19S allele is a marker for hyponatremia in the study cohorts. It was hypothesized that the variant channel would be less responsive to hypotonicity in vitro; decreased sensitivity of a hypotonicity sensor in vivo would be permissive with respect to water excess. Therefore, the impact of the TRPV4^P19S polymorphism was functionally evaluated in a heterologous expression system. First, the subcellular distribution of TRPV4^WT and TRPV4^P19S was tested by confocal immunofluorescence microscopy in HEK293 cells transiently transfected with a cDNA coding for full-length wild-type human TRPV4, or with a cDNA mutated to incorporate the TRPV4^P19S polymorphism. TRPV4^WT and TRPV4^P19S showed similar levels of expression and localization to the plasma membrane, as determined via confocal immunofluorescence microscopy (Figure 3) and via cell surface biotinylation experiments (Figure 4) . No TRPV4 was immunodetectable in HEK293 cells transfected only with GFP (Fig. 5) .

Whole-cell currents were recorded in HEK293 cells transiently transfected with a cDNA coding for full- length wild-type human TRPV4, or with a cDNA mutated to incorporate the TRPV4^P19S minor allele. Currents in response to a mild hypotonic stressor (corresponding to a 15% reduction in osmolality) were markedly diminished in the TRPV4^P19S transfectants relative to cells transfected with the wild-type TRPV4 (Fig. 2A, 2B), whereas the response to the synthetic TRPV4 agonist 4α- phorbol 12, 13-didecanoate (4αPDD) was unaffected by the polymorphism (Figures 2A, 2B) . Plots of current-voltage relationship obtained at the indicated time points in Figures 2A and 2B are shown in Figures 2C and 2D, respectively. Whole cell cationic currents activated by 4αPDD (in TRPV4^WT and TRPV4^P19S expressing cells) and 15% hypotonicity (in TRPV4^WT cells) presented outward rectification similar to that previously described for TRPV4 (Strotmann et al . (2000) Nat. Cell Biol., 2:695- 702; Watanabe et al. (2002) J. Biol. Chem. , 277:13569- 77; Arniges et al. (2006) J. Biol. Chem., 281:1580-6) . In aggregated data, mean current density in response to the mild hypotonic stress was significantly less in the TRPV4^P19S variant allele than in wild-type TRPV4 (Fig. 2E) . Interestingly, the responses to a more pronounced degree of hypotonicity (corresponding to a 30% reduction in medium osmolality; Fig. 2E) did not differ significantly between the two alleles. Exposure to 30% hypotonic solution did not activate significant whole- cell cationic currents in GFP-transfected HEK293 cells (Fig. 6) . Importantly, the TRPV4^P19S allele exerted a dominant negative effect with respect to the wild-type allele. When cotransfected in a 1:1 ratio (i.e., mimicking a human subject heterozygous for the TRPV4^P19S allele of this autosomal gene) , the variant allele suppressed the response of the wild-type allele to 15% hypotonicity (Fig. 2E) . TRPV4^P19S channels also showed a decreased response to the osmotransducing messenger, epoxyeicosatrienoic acid (156 nM/ Fig. 2F) . These data indicate that the TRPV4^P19S allele codes for a variant channel that exhibits reduced responsiveness to mild hypotonic stress (i.e., such as that likely to be encountered in vivo) and to the intracellular lipid messenger, epoxyeicosatrienoic acid, but not to more pronounced hypotonic stress or to 4αPDD.

EXAMPLE 2

A panel of hyponatremic subjects was screened and another polymorphism affecting the same region of the TRPV4 molecule as the TRPV4^pl9s polymorphism was identified. This polymorphism, TRPV4^A1OP, was not present in any publicly curated database and coded for an alanine-to-proline amino acid change at residue number 10 of the TRPV4 protein (more specifically, it represented a G>C change in the first nucleotide of the codon) . This polymorphism gave rise to a CCG rather than GCG codon coding for Proline-10 in the TRPV4 protein sequence. This polymorphism occurred in conjunction with (i.e., in linkage disequilibrium with) three adjacent polymorphisms: 1) OT in the third nucleotide of the codon coding for amino acid Pro-19 (this was a synonymous polymorphism because it affected the third nucleotide of the codon and did not result in a change in amino acid); 2) T>C change in the third nucleotide of the codon coding for amino acid Gly-27 (this was also a synonymous polymorphism and did not result in a change in amino acid) ; and 3) OT change in the 5' untranslated region of the TRPV4 cDNA twenty base-pairs upstream of the ATG consensus start site for TRPV4 (changing this potential regulatory region from tgagcagtgCagacgggcctggggcaggcATG (SEQ ID NO: 5) to tgagcagtgTagacgggcctggggcaggcATG (SEQ ID NO: 6)), where ATG indicates the putative translational start site.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A method of diagnosing an increased risk for hyponatremia in a subject, said method comprising: a) obtaining a biological sample from said patient; and b) assessing the presence of a mutant transient receptor potential-vanilloid-4 (TRPV4), wherein the presence of a mutant TRPV4 indicates said patient has an increased risk for hyponatremia.

2. The method of claim 1, wherein said mutant TRPV4 is hypofunctioning .

3. The method of claims 1, w.herein step b) comprises assessing the presence of a residue other than proline at position 19 of TRPV4 and/or the presence of a residue other than alanine at position 10 of TRPV4.

4. The method of claims 3, wherein step b) comprises assessing the presence of a residue other than proline at position 19 of TRPV4.

5. The method of claim 4, wherein said residue at position 19 is a non-conservative change from proline.

6. The method of claim 4, wherein said residue at position 19 is serine.

7. The method of claims 3, wherein step b) comprises assessing the presence of a residue other than alanine at position 10 of TRPV4.

8. The method of claim 7, wherein said residue is a non- conservative change from alanine.

9. The method of claim 7, wherein said residue is proline .

10. The method of claim 1, wherein said TRPV4 has at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2.

11. The method of claim 1, wherein step b) comprises sequencing a nucleic acid molecule encoding said TRPV4.

12. The method of claim 1, wherein said subject has environmental risk factors for the development of hyponatremia .

13. The method of claim 12, wherein said subject is elderly, engages in heavy activity, or takes medications which may cause hyponatremia.