A Nofel Homozygous KLHL3 Treiglad A Achos O Awtomal Recessive Pseudohypoaldosteronism Math II Diagnosis Hwyr yn Bywyd Ⅱ
Nov 02, 2023
Achos Adroddiad
The subject, a female of Turkish origin, was referred at the age of 58 years to the outpatient clinic of the Department of Nephrology, University Hospital Düsseldorf, for persistent hyperkalemia (first diagnosed at age 47 years), suspected type IV renal tubular acidosis in the presence of chronic kidney disease (CKD) (G3a A2, KDIGO), cirrhosis of the left kidney, nephrolithiasis with a history of surgical stone removal at age 17 years, recurrent urinary tract infections, and hypertension (first diagnosed at the age of 41 years). Her past medical history was significant for coronary heart disease, with status post-non-ST segment elevation myocardial infarction at the age of 47 years, multiple stent implantations, and ST-elevation myocardial infarction 5 months prior to referral. After the second myocardial infarction, serum potassium levels had risen up to 7.3 mmol/L, and severe muscle pain had started. The patient also reported paroxysmal atrial fibrillation, hypercholesterolemia, and allergy to contrast agents. Medications included bisoprolol 2.5 mg bid, calcium polystyrene sulfonate 15 g bid, sodium hydrogen carbonate 3 g tid, phenprocoumon, atorvastatin 40 mg qd, aspirin 100 mg qd, pantoprazole 40 mg qd, L-thyroxin 25 ug qd, and clopidogrel 75 mg qd. Temporary discontinuation of statin therapy had not alleviated muscle pain. Discontinuation of an AT1 receptor antagonist that had been on her regimen as well as a diet low in potassium had not led to an improvement of hyperkalemia.
Family history was significant for parental consanguinity (I degree cousins), hypertension in both parents, a fatal stroke in her mother in her early 60s, and coronary artery disease in her father. Her brother had died from renal disease of unclear origin in Turkey in his 50s (shown in Fig. 1 a).
At the amser gwerthuso, gwaed pwysedd was 148/87 mm Hg, a her calon cyfradd roedd 60% 2fmin. Clinigol archwiliad was anremarkable, apart o edema o y isel eithafion, lluosrif hematomas, a striae distensae.
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Labordy arholiad arddangos hyperkalemia (serwm potasiwm o 5.0.00 3 mmol/L [normal 3.6–4.8]), arferol serwm sodiwm (139 mmol/L, (arferol 135–145), hyperchloremia (107 mmol/L, normal 95–105), hypomagnesemia yn 0.55 mmol/L (arferol 0.73–1.00}), serum creatinine at 1.18 mg/dL (normal <0.90) with an estimated glomerular filtration rate of 51 mL/min (CKD-EPI [40], normal 90–140), elevated urea of 47 mg/dL (normal 21– 43), suppressed renin concentration (<1.0 pg/mL [normal 1.7–23.9]), and aldosterone of 157 pg/mL (normal 12–236). Serum calcium and phosphate were normal (2.31 mmol/L [normal 2.10–2.42] and 0.95 mmol/L [normal 0.84–1.45], respectively). Venous blood gas analysis revealed pH 7.33 (normal 7.35–7.43), standard bicarbonate 19.8 mmol/L (normal 24–30), and standard base excess −5.3 mmol/L (normal −2 to +3). Spot urinary potassium was low (6.5 mmol/L [normal 10–200]), whereas spot urinary calcium was normal (0.36 mmol/L); urinary creatinine was 24.2 mg/dL. There was no detectable albuminuria or proteinuria.
Renal uwchsain wedi'i ddangos a bach hawl arennau (8.5 cm modfedd yn hyd) gyda thin, inhomogeneous parenchyma, a 12-mm wedi'i diffinio'n dda rownd cyst. Y chwith arenni roedd arferol yn maint (10 cm modfedd hyd) , gyda normal parenchymal trwch, A wedi'i diffinio'n dda�% A0Round Cywasgu A Cymhleth {}}}mm wedi'i diffinio'n dda Rownd Cyst gyda amau septwm. Roedd Tystiolaeth�% A0of hydronephrosis.
Y presenoldeb o hyperkalemic metabolig asidosis gyda yn annigonol isel wrinol potasiwm ysgarthu, a oedd esbonio esbonio by CKD alone, isel-renin pwysedd gwaed gyda aldosterone bod yn yn annigonol isel yn ar gyfer hyperkalemia, a parental consanguinity led us i consider PHA II. Cychwyn o therapi gyda hydrochlorothiazide 25 mg qd led i A gostyngiad o serum potasiwm i 4.2 mmol/L o fewn 1 wythnos, a gwaed pwysedd normaleiddio. Calsiwm polystyren sylffon a sodiwm hydrogen carbonad yn cyflym dod i ben. A dros dro gostyngiad o hydrochlorothiazide i 12.5 mg qd led i'r ailymddangosiad o hyperkalemic asidosis. Hyponatremia digwydd ar ôl hydrochlorothiazide therapi a datrys ar diet yn halen. Dau mis ar ôl y ymweliad cyntaf ymweliad i ein clinig, y adroddwyd i ddechrau cyhyrau poen had datrys. Minor poen bod oedd disgrifio fel gwahanol cymeriad was briodoli to statin therapi yn y presenoldeb o ysgafn CK (153 U/L, normal <145) and LDH (250 U/L, normal <147) elevation. A review of prior therapy for hypertension revealed a regimen of valsartan plus hydrochlorothiazide (12.5 mg) prior to myocardial infarction, after which hydrochlorothiazide had been discontinued, apparently unmasking electrolyte abnormalities and causing muscle pain.
Genetig Dadansoddi
Because of the clinical suspicion of PHA II, we performed PCR and Sanger sequencing of the KLHL3, CUL3, WNK1, and WNK4 genes (see Methods). No variants in the WNK1, WNK4, and CUL3 genes were identified. However, a homozygous chr5:g.136973013 G > A (GRCh37) variant was identified in the KLHL3 gene, resulting in a p.Arg431Trp missense mutation (NP_059111.2) predicted as probably damaging by PolyPhen [41], deleterious by SIFT [42] and disease-causing by MutationTaster [43] (shown in Fig. 1b). This variant is present once in a heterozygous state among 251,436 alleles in gnomAD (allele frequency 4 × 10−6) [44] and once in a heterozygous state among 21,366 alleles in the ALFA Project (www.ncbi.nlm. nih.gov/snp/docs/gsr/alfa/; allele frequency 5 × 10−5). It is listed in dbSNP as rs769995865 and has not been reported in ClinVar. Arginine at human position 431 is completely conserved among 100 vertebrates (http://genome.ucsc. edu/cgi-bin/hgTrackUi?db=hg19&g=multiz100way, with the exception of horse, for which no sequence is available), in C. intestinalis (vase tunicate), D. melanogaster (fruitfly), and C. elegans (nematode worm) (shown in Fig. 1c). Out of 41 paralogous proteins, arginine is conserved in 17, with none of the paralogs showing tryptophan at this position (online suppl. Fig. 1). The finding of a homozygous variant in the setting of consanguinity, in combination with its rarity in the general population and the evolutionary conservation of the affected residue, led us to consider the KLHL3 p.Arg431Trp mutation as a candidate for disease causation. The variant is located in the Kelch-like repeat 3 (shown in Fig. 1d). A homozygous p.Arg431Gln variant at the identical residue has been previously described in a single individual [12] (Table 1).
Strwythurol Dadansoddi
The Kelch domain (shown in Fig. 2a) is composed of six "propeller blades" stabilized by inter-blade hydrogen bonds [45]. Previous simulation studies of Kelch-domain surface mutations demonstrated decreased interaction with the WNK4 peptide but found little disruption of Kelch-WNK4 binding by mutations in the core of the protein [46]. Here, we used equilibrium molecular dynamics simulations to determine the structural and dynamic consequences of the R431W core mutation. R431W is positioned at the interface between the third (K3) and fourth (K4) propeller blades, situated directly underneath the WNK4-binding site (shown in Fig. 2a). Calculation of the difference in mean squared fluctuations (a measure of variance in the protein structure) between R431W and WT simulations reproducibly demonstrated that the perturbation in protein dynamics caused by the mutation propagates throughout the entire protein, with strongly increased structural dynamics at the K3–K4 interface and the surrounding K3–K5 region, but also decreased dynamics at more distant sites (shown in Fig. 2b). Global RMSD calculations indicated a slight increase in structural instability of the mutant over the 1.1-µs simulations – focused RMSD calculations of the K3–K4 interface (residues 425–465) and the WNK4-binding site (residues 339, 355, 360, 386, 402, 407, 432, 449, 451, 481, 498, 528, and 577), suggesting a disruption of tertiary structure in the R431W mutant (shown in Fig. 2c, e; see below).
Ar gyfer pellach meintioli o y rhyngweithio rhwng y K3 (gweddillion 425–441) and K4 (gweddillion 442–465) llafnau, y rhif o hydrogen bondiau (h-bonds) a y y COM pellter rhwng y dau grwpiau were cyfrifedig. Y WT rhyngwyneb maintains, ar ar cyfartaledd, 7 mwy hbonds na yn yn y R431W mutant. R431 itself only cymryd rhan yn 4% e2��5 h-bondiau – accounting ar gyfer ∼27% of y K3–K4 h-bondiau – indicating that y R431W treiglad tarfu y entire rhyngwyneb (dangos yn Fig. 2d, e). At , y R431W arddangosfeydd an cynyddodd COM pellter rhwng y dau is-barthau (dangos mewn Fig. 2d), cyrydu y h-bond dadansoddiad.
I assess yr effeithiau o y treiglad i WNK4 rhwymo, ni cyfrifedig y electrostatig o y rhwymo poced throughout the 1.{{2}µs efelychiad. Y R431W treiglad cymell a cymedrol newid yn y rhwymo-poced electrostatig tuag negatif foltedd (dangos yn Ffig.% 2d). Er modest, y cadarnhaol potensial yn y rhwymo negyddol motiff .
Swyddogaethol Dadansoddiad
To further study the pathophysiology associated with KLHL3 p.Arg431Trp, we obtained plasmids encoding 3x FLAG-tagged human WNK4 in the pTRE2hyg vector and Halo-tagged human KLHL3 in the pFN21A vector [47]. We introduced the p.Arg431Trp mutation to the WT KLHL3 plasmid using site-directed mutagenesis and heterologously expressed the corresponding plasmids in COS7 cells. When coexpressing WT and mutant KLHL3 with WT WNK4, Western blots with anti-Halotag primary antibodies revealed WT KLHL3 bands. However, repeated transfections of two independent KLHL3 p.Arg431Trp clones initially did not result in detectable bands (relative intensity normalized by -actin expression, 0.761 ± 0.144 in WT vs. 0.0{{3{{40}}}}9 ± 0.002 in p.Arg431Trp, p < 0.0001, t=12.81). Expression levels of WNK4, determined by anti-FLAG Western blot, were significantly higher in cells coexpressing KLHL3 p.Arg431Trp than in cells coexpressing WT KLHL3 (relative intensity normalized by -actin expression, 0.721 ± 0.137 with WT vs. 0.967 ± 0.115 with Arg431Trp, p=0.005, t=3.546). All values are given as mean ± SEM, and comparison was performed by a two-tailed ratio-paired t test, df=11 (shown in Fig. 3a, b). To assess whether KLHL3 p.Arg431Trp was expressed at lower quantities, we transfected increasing amounts (0, 0.5, 1, 2, 4, and 8 µg) of WT and mutant plasmids. With 8 µg, we observed increased lethality (data not shown), but with expression of 4 µg mutant plasmid, we could detect a weak band at the expected size (note the longer exposure time compared to WT, shown in Fig. 3c). To assess whether decreased protein stability could cause decreased expression levels of the mutant protein, we performed cycloheximide chase assays. KLHL3 p.Arg431Trp was degraded more rapidly than WT (shown in Fig. 3d).
Fig. 3. A Representative Western blots of cell lysates transfected with WNK4 and empty vector, WT or p.Arg431Trp (R431W) KLHL3. b Box and whisker plots (Tukey) with quantitation of the results from 12 experiments, using two independent clones of each plasmid. **, p=0.0046; ****, p < 0.0001 (two-tailed ratio-paired t tests). c Representative Western blots of cell lysates transfected with increasing amounts of WT or R431W KLHL3. Note the different exposure times for WT and R431W. The lower and upper HaloTag-positive bands may reflect the KLHL3 monomer and dimer, respectively. d Representative Western blots of cells transfected with 2 µg WT or 4 µg R431W KLHL3 plasmid, treated with cycloheximide and lysed at the indicated time points. Note the different exposure times. Analysis of 2 biological replicates with 2 technical replicates each shows decreased stability of mutant protein, normalized to beta-actin and time point 0 h (mean ± SD). At 12 h, significantly less R431W than WT protein is present (Mann-Whitney test, *, p=0.029).
Trafodaeth/Casgliad
At the age of 58 years, the patient reported here was diagnosed later in life than any of the previously reported cases with recessive KLHL3 mutation. The oldest patient reported in the literature was diagnosed at the age of 56 years [15]. However, retrospectively, hypertension and hyperkalemia had been present in our patient for several years. The characteristic hyperkalemic metabolic acidosis associated with PHA II had likely been masked by the administration of a thiazide diuretic. Discontinuation of hydrochlorothiazide after myocardial infarction was the likely cause of massive hyperkalemic acidosis associated with muscle pain and hypertension that eventually led to the diagnosis of PHA II. We thus suggest that the presence of hyperkalemic metabolic acidosis that is inadequate for kidney function in combination with suppressed renin should raise the suspicion of PHA II even in patients over 50 years of age. The diagnosis of PHA II can be complicated by the effects of preexisting treatment with antihypertensive medications that influence the renin-angiotensin-aldosterone system and may lead to spuriously elevated or low renin and/or aldosterone values [48, 49]. Hyperkalemia despite a normal glomerular filtration rate is often described as a characteristic feature of PHA II [6]. While this may be characteristic in children, in adults, such as in the patient reported here, longstanding hypertension, nephrolithiasis, and recurrent urinary tract infections can lead to impaired kidney function, as previously reported in a patient with a heterozygous mutation [14]. Nephrolithiasis as in our patient has been described in PHA II [50] but is not a common finding. Interestingly, spot urinary calcium: creatinine ratios were not elevated in our patient (0.17 and <0.06 mmol/mmol creatinine, respectively), contrary to observations in large kindred with the WNK4 mutation and average urinary calcium of 0.85 ± 0.27 mmol/mmol creatinine [51]. Severe target-organ damage in our patient, including myocardial infarctions and CKD, emphasizes the need for early diagnosis and adequate therapy of PHA II, although hypertension and target-organ damage in our patient's parents may also point to a genetic risk independent of the homozygous PHA II mutation. To prevent delayed diagnosis, after more common causes, such as primary aldosteronism, have been excluded, genetic testing for Mendelian forms of hypertension should be considered by practitioners when patients present with low-renin hypertension in the presence of hormonal and/or electrolyte abnormalities.
Samplau o 'r y claf's teulu oedd nid ar gael ar gyfer dadansoddi; however, yn y lleoliad o cysondeb, mae'n yn yn tebygol bod y p.Arg431Trp treiglad . gwybodaeth ystyried�% a0the patient's ymadawedig brawd oedd nid ar gael. Mae'n conceivable hynny he, too, dioddef o PHA II, canlyniad yn yn CKD a oedd yn y pen draw angheuol.
It is possible that the p.Arg431Trp mutation observed in our patient prevents the Kelch domain from folding, consistent with the decreased structural stability of the mutated domain observed in our simulations, where the side-chain substitution was inserted in the already folded protein. Accordingly, Western blot analysis indicated significantly reduced expression levels of p.Arg431Trp KLHL3 (shown in Fig. 3a-c). The cycloheximide (an inhibitor of protein biosynthesis) chase assay further demonstrated reduced protein stability of p.Arg431Trp KLHL3 (shown in Fig. 3d), similar to prior observations in the p.Ser410Leu variant [52]. These data indicate that the primary consequence of the p.Arg431Trp mutation is to destabilize the folded state of the protein, whereas de-creased interactions of folded KLHL3 with WNK4 is, at most, a secondary effect. In any case, lower levels of KLHL3 reduce WNK ubiquitinylation, resulting in higher WNK4 abundance (shown in Fig. 3) and are inferred to cause higher NCCT phosphorylation, as well as lower potassium secretion via ROMK, explaining the underlying pathophysiology.
Cydnabyddiaeth�% a0
Rydym diolch Dr. Shinichi Uchida (Tokyo Meddygol a Prifysgol Dental ) ar gyfer kindly darparu y pTRE2hyg WNK4 a pFN21A KLHL3 plasmids, Iana Lukianova am cefnogaeth gyda Gorllewinern blot arbrofion, a Gabriel Stölting for helpful discussions.
Datganiad o Moeseg
Mae'r ymchwil protocol was approved by the Ethics Committee o the Medical Faculty, Heinrich Heine University Düsseldorf (astudiaeth rhif 4330) , a ysgrifenedig gwybodaeth cydymffurfiad gyda y Helsinki Datganiad was a gafwyd o y ymchwil cyfranogwr for yr ymchwil astudiaeth a cyhoeddiad o hwn achos adroddiad.
A.E., N.H., B.G.H., J.P.M., a T.M. wedi gwrthdaro o o diddordeb to dedatganre. L.C.R. received personal ffioedd ar gyfer ymgynghoriaethau a siarad ymrwymiadau o Astellas, Baxter, Bayer-Vital, Boehringer, Fresenius, Medtronic, Novartis, a Recor, gyda rhif perthnasedd�% A0to papur U.I.S. is rhestredig as ar patentau UD 10% 2c696,739 a 16% 2f614% 2c401% 2C gyda Rhif Perthnasedd i y papur.
Cyllid Ffynonellau�% a0
Mae hyn astudiaeth was ariannwyd gan y Ministerium für Kultur und Wissenschaft der Landes Nordrhein-Westfalen (Rückkehrprogramm), y Stiftung Charité (BIH_}}}}}), a the German Ymchwil Foundation (DFG, SCHO 1386/2-1; prosiect-ID 431984000, CRC 1453), all i U.I.S. It Roedd ariannwyd gan y Deutsche Forschungsge meinschaft (Almaeneg Ymchwil ) i J. P (MA 7525/2- 1, fel rhan o y ymchwil uned FOR 5046, prosiect P2) and by grant o o y Interdisciplinary Canolfan ar gyfer Clinigol Ymchwil o fewn cyfadran o Meddygaeth yn y RWTH Prifysgol (IZKF TN1- 3/IA532003). Mae'r awduron ddiolchgar cydnabyddu y cyfrifiadura amser a ganiatawyd trwy JARA ar uwchgyfrifiadur JURECA yn Forschungszentrum Jülich. Funding agencies no rôl yn yn y yn y paratoi o y llawysgrif.
Cyfeirnodau
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