Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting

Loss-of-function mutations of SLC34A3 represent an established cause of a distinct renal phosphate wasting disorder termed hereditary hypophosphatemic rickets with hypercalciuria (HHRH). SLC34A3 encodes the renal phosphate transporter NaPi2c expressed at the apical brush border of proximal renal tubules. Substitution of p.Ser192Leu is one of the most frequent genetic changes among HHRH patients in Europe, but has never been systematically evaluated, clinically or on a cellular level. Identification of a 32-year-old female with a homozgyous c.575C>T, p.Ser192Leu substitution enabled a more comprehensive assessment of the impact of this missense variant. Clinically, the patient showed renal phosphate wasting and nephrocalcinosis without any bone abnormalities. Heterozygous carriers of deleterious SLC34A3 variants were previously described to harbor an increased risk of kidney stone formation and renal calcification. We hence examined the frequency of p.Ser192Leu variants in our adult kidney stone cohort and compared the results to clinical findings of previously published cases of both mono- and biallelic p.Ser192Leu changes. On a cellular level, p.Ser192Leu-mutated transporters localize to the plasma membrane in different cellular systems, but lead to significantly reduced transport activity of inorganic phosphate upon overexpression in Xenopus oocytes. Despite the reduced function in ectopic cellular systems, the clinical consequences of p.Ser192Leu may appear relatively mild, at least in our index patient, and can potentially be missed in clinical practice. Electronic supplementary material The online version of this article (10.1007/s00240-019-01116-2) contains supplementary material, which is available to authorized users.


Introduction
Tight regulation of serum phosphate is crucial for various cellular processes, such as maintenance of energy balance, prevention of vascular calcification, bone metabolism, and acid-base homeostasis [1]. Driven by the phosphaturic effects of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23), the kidneys play an important Ria Schönauer, Friederike Petzold contributed equally to this work.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s0024 0-019-01116 -2) contains supplementary material, which is available to authorized users. 1 3 role in the excretion of excess phosphate from the circulation. The phosphaturic effect is achieved by inhibition of renal phosphate reabsorption from the primary urine via two Na + -dependent phosphate transporters NaPi2a/ SLC34A1 and NaPi2c/SLC34A3 [2]. On the other hand, low serum phosphate triggers vitamin D-dependent enteral calcium reabsorption and may lead to hypercalcemia and hypercalciuria.
Hypercalciuria is the most common metabolic finding among kidney stone formers [3]. Its genetic basis, however, is poorly understood. Monogenic kidney stone disorders shed light on mechanisms of hypercalciuria that may also pertain to the general population of kidney stone formers [4]. One of these disorders is hereditary hypophosphatemic rickets with hypercalciuria (HHRH; OMIM #241,530) characterized by renal phosphate wasting and elevated calcium levels in blood and urine. In 1987, Tieder et al. first described isolated hypercalciuria and HHRH as two phenotypic expressions of a shared underlying defect in an extended Bedouin kindred [5]. It took another 19 years until two groups independently deciphered loss-of-function mutations of the proximal renal tubular phosphate transporter SLC34A3/NaPi2c as the underlying genetic defect [6,7]. The electroneutral transporter NaPi2c, like its electrogenic paralog NaPi2a/SLC34A1, is expressed at the luminal brush border of proximal renal tubular cells, responsible for finetuning phosphate reabsorption from primary urine to the basolateral bloodstream. Malfunction or loss of function of NaPi2c results in urinary phosphate wasting, hypophosphatemia and a consecutive increase in systemic 1,25-(OH) 2 D levels [8]. Among several mutations identified, both initial genetic studies found a heterozygous missense variant at amino acid residue 192 changing serine to leucine as part of compound heterozygous SLC34A3 mutations [6,7]. Although the p.Ser192Leu variant has been reported several times in patients with HHRH and isolated hypercalciuria, it has never been systematically evaluated with regard to clinical or molecular pathogenicity. In recessive disorders homozygous mutations are helpful to assess the clinical impact of a certain genetic variant as exemplified here by a young adult with nephrocalcinosis due to a homozygous SLC34A3 c.575C > T, p.Ser192Leu mutation. We provide an evaluation of the mutation's clinical and molecular impact and raise the question whether p.Ser192Leu may be enriched in idiopathic kidney stone formers.

Patient cohort
The index patient was recruited for genetic analysis after informed consent and enrollment in our clinical registry for hereditary kidney stone disorders at the University of Leipzig (Germany) [9]. Patients with idiopathic kidney stone disease were tested for genetic causes after written informed consent and prior study approval by the Institutional Review Board (IRB) of the Universities of Leipzig and Newcastle. The cohort comprised 670 patients, 61% males and 35% females, with a median age of 50 years (range 1-88 years) and a median age at first stone event of 36 years (range 0-86 years).

Expression vectors
The vector pEGFP_C1_NaPi2c_WT [10] for the expression of a fusion protein of N-terminal enhanced green fluorescent protein (eGFP) connected by a linker sequence (SGLRS-RAQASNS) to wild-type NaPi2c was constructed by ligation of the human SLC34A3-sequence (NM_001177316.1) into the pEGFP-C1 vector backbone using EcoRI and SacII restriction sites that were added by mutagenesis [7]. To establish the mutant plasmid pEGFP_C1_NaPi2c_S192L, the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Massachusetts, USA) and custom-designed back-to-back primers (NaPi2c_S192L_Fw: 5′-GCT TTC AGC GGC TTG GCG GTG CAC -3′; NaPi2c_S192L_Rv: 5′-CCT CTG AAA TTC ATC CCG GTC CCC TG-3′) were used to introduce the base exchange 575C>T into pEGFP_ C1_NaPi2c_WT according to the manufacturer's instructions. Correctness of all sequences was verified by Sanger sequencing.

Molecular biology oocyte injections
Constructs for oocyte injections were generated by PCR adding a T7 RNA transcription start site and a short poly-A tail using mammalian SLC34A3 expression plasmids as templates [10]. The primer sequences are: T7 -NaPi2c, 5′-AGC CAC TAA TAC GAC TCA CTA TAG GGC TCC GCT GCC ACC ATG CCG AGT TCC CTT CC-3′; T7 -GFP-NaPi2c, 5′-AGC CAC TAA TAC GAC TCA CTA TAG GGC TCC GCT GCC ACC ATG GTG AGC AAG GGC GAG-3′ and SV40 pA terminator, 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TCA AAA TAT TAA CGC TTA CAA TTT ACG CG-3′. 0.1 µg of the purified DNA fragments was in vitro transcribed using the T7 mMessageMachine kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 10 ng of RNA was injected per oocyte.

Xenopus laevis oocytes
Oocytes were purchased from Ecocyte (Dortmund, Germany) and established protocols for maintenance and injections were followed [11]. Oocytes were assayed after 3 days measuring fluorescence and uptake of [ 32 P] inorganic phosphate. Fluorescence at the oocyte surface was measured using an inverted fluorescence microscope (Nikon, Tokyo, Japan) with identical settings for each measurement and quantified using image FIJI.

Results
In a 32-year-old female (index patient, II1) with nephrocalcinosis on renal ultrasound scanning, we observed mild hypophosphatemia, hypercalcemic hypercalciuria, and reduced tubular phosphate reabsorption. Vitamin D3, PTH, and FGF23 levels were within the reference range (Table 1; Fig. 1a). The patient was of normal height (1.70 m) and weight (55 kg) (BMI 19.0 kg/m 2 ) and did not report any prior kidney stone passages. Past medical history was negative for fractures or bone abnormalities and family history was completely negative for both, renal and bone disease. Upon targeted next-generation sequencing of known OMIM causes of nephrocalcinosis and nephrolithiasis (including ATP6V1B1, ATP6V0A1, CLCN5, CLDN16, CLDN19, CYP24A1, PHEX, SLC34A3, SLC22A12, SLC2A9, SLC34A1, SLC3A1, SLC4A1, SLC7A9, SLC9A3R1), we identified a homozygous c.575C>T, p.Ser192Leu mutation in SLC34A3 as underlying genetic diagnosis (NM_001177316) (Fig. 2a, b). Segregation analysis confirmed heterozygous Ser192Leu variants in the parents (I1, I2) as well as in the younger brother (II2) of the index patient (Fig. 2a, b). As part of retrospective phenotyping, we undertook dual X-ray absorptiometry (DXA) and bone scan in the index patient. Both imaging techniques showed no abnormalities with regard to bone mineral density and technetium uptake, indicating normal bone metabolism (Fig. 1b-d). Further clinical assessment included biochemical analysis and renal ultrasound scanning of the heterozygous family members. Serum parameters revealed normal values; however, minimal signs of renal calcifications were seen in both parents and the brother ( Table 1; Fig. 1a). In contrast to the only other previously reported individual (E/II-2) carrying a homozygous p.Ser192Leu mutation (Table 1), our index patient did not exhibit nephrolithiasis as a child and kidney function remained preserved despite renal calcifications. Upon daily oral phosphate supplementation (1800 mg/day), metabolic abnormalities of hypercalcemic hypercalciuria and hypophosphatemic hyperphosphaturia normalized completely within several weeks (Suppl. Table 1).
In a next step, we sought to evaluate the frequency of p.Ser192Leu in a cohort of heterogeneous idiopathic adult kidney stone formers with or without hypercalciuria. Upon genetic analysis of 670 individuals with various types of kidney stones, we identified two more patients with heterozygous SLC34A3 c.575C>T, p.Ser192Leu mutations. Both patients presented with a history of recurrent kidney stone events, but unspecific metabolic parameters and absence of renal calcification and overt bone manifestations (Table 1; N77/CSS1162 and N137/CSS1355).
To investigate the functional impact of the NaPi2c p.Ser192Leu mutation on a cellular level, we first performed overexpression of GFP-tagged mutant (Ser192Leu) and wild-type (WT) NaPi2c in HEK293 cells. While both constructs correctly localized to the plasma membrane, cells transfected with the mutant construct showed a slightly reduced expression on the cell surface in comparison to the wild type (Fig. 3a). Similarly, we injected both constructs into Xenopus laevis oocytes to test for membrane localization and functional P i uptake. In line with the expression in HEK293, the mutant transporter was efficiently sorted to the plasma membrane (Fig. 3b). Upon oocyte 32 P i -uptake, injected GFP and non-GFP mutants showed a significant reduction in transport activity in comparison to wild-type RNA (Fig. 3c). On the other hand, co-injection of WT and mutant RNA did not lead to a significant reduction of 32 P i -uptake. To conclude, the p.Ser192Leu mutation does not have a significant impact on membrane sorting of NaPi2c but severely reduces its transport activity.

Discussion
Evaluating the pathogenicity of genetic variants is crucial in the era of accelerated genetic diagnostics. To date, only a minority of the overall 38 reported HHRH-associated SLC34A3 mutations (HGMD Ⓡ professional version 2018.3) have been studied functionally [7,10,13]. Although p.Ser192Leu is one of the few repetitive findings in HHRH patients, it has never been assessed for its pathogenicity on a cellular level. Based on its high sequence similarity with NaPi2b, the electroneutral P i transporter NaPi2c is assumed to harbor six transmembrane domains [14] and two inverted core domains that move during the translocation process. Ser192 represents a highly conserved amino acid, most likely located within the third transmembrane domain adjacent to the substrate binding site (Figs. 2c, 4) [15,16]. Human Ser192 is part of the previously described SSG/AAD motif (p.Ser189Ala, p.Ser191Ala, and p.Gly195Asp; corresponding to murine NaPi sequences) that is involved in binding of the first Na + ion and affects the electrogenicity of co-transporter function [17]. A change of the adjacent Gly196 residue to arginine was also shown to result in trafficking defects with endoplasmatic reticulum retention and absence of membrane localization [13,17]. Interestingly, unlike p.Gly196Arg, p.Ser192Leu seems to be adequately processed on its way to the cell surface, but lacks transport activity when expressed at the luminal brush border. P.Ser192Leu occurs in the general population as a rare genetic variant in about 0.05% of individuals worldwide. With an allele frequency of 0.1%, p.Ser192Leu is reported almost exclusively in individuals of European, non-Finnish descent (86/87.762). Homozygosity, however, is not listed in SNP databases of the general population (ExAc/ gnomAD). Although statistically not significant, our finding of two additional heterozygous p.Ser192Leu carriers   [7] in a population of 670 kidney stone formers suggests that p.Ser192Leu may be enriched in common nephrolithiasis (0.3%). This is in line with genome-wide association studies (GWAS) that reported a contribution of genetic variants within Mendelian nephrolithiasis genes to general kidney stone susceptibility [4]. Recently, Dasgupta et al. reported a  significantly increased risk of renal calcification not only in patients with biallelic mutations, but also among heterozygous SLC34A3 mutation carriers. In our functional approach to test for pathogenicity of heterozygous p.Ser192Leu, we did not identify a clear dominant influence of the mutated transporter on wild-type protein function. Similarly, in the family presented here, heterozygous carriers also showed no overt clinical manifestation, but slight biochemical abnormalities. These findings indicate a very mild effect of heterozygous mutations that may only become symptomatic with additional disease triggers, such as dehydration, lithogenic diet or a particular genetic background. Homozygosity of p.Ser192Leu in our patient is also associated with a very mild clinical phenotype without impairment of renal function and bone manifestations. Therefore, p.Ser192Leu, although clearly pathogenic upon expression studies, may be clinically compensated in some patients. In summary, we report for the first time the deleterious effect of NaPi2cp.Ser192Leu by clinical presentation of a patient homozygous for the mutation and demonstrate its negative impact on P i transport activity despite adequate membrane localization. In contrast to the evident molecular pathogenicity, the clinical picture of p.Ser192Leu may appear relatively mild and might therefore be overlooked in clinical practice. However, as demonstrated by the slightly differential phenotype of the only other reported individual with a homozygous p.Ser192Leu mutation (E/II-2- Table 1), clinical appearance is obviously influenced by individual environmental and/or genetic modifiers. Fig. 3 Functional evaluation of NaPi2c-Ser192Leu in comparison to wild type. a Plasma membrane localization of GFP-tagged mutant (Ser192Leu) and wild-type (WT)-NaPi2c proteins upon overexpression in HEK293 cells. b Fluorescence intensity, indicating plasma membrane localization, of negative control, positive control, NaPi2c mutant (Ser192Leu), and NaPi2c wild type (WT) upon Xenopus oocyte injection shows no significant difference. c 32 P i -uptake upon oocyte injection of NaPi2c mutant (Ser192Leu) is significantly disturbed in comparison to WT in both, GFP-tagged and non-GFP tagged conditions

Fig. 4
Localization and predicted topology of SLC34A3/NaPi2c wild type and mutant. a Cartoon of NaPi2c with its renal localization at the brush border of promixal tubules. The hypothetical structure based on sequence similarity with NaPi2b consists of six transmembrane domains (TM), where Ser192 (red) is part of the third trans-membrane helix (light gray), adjacent to the substrate binding site. b Model of flNaPi2b (PM0080462) [15] showing NaPi2c Ser192 (red) (corresponding to NaPi2b Ala192) localization within the third transmembrane (TM) helix (light gray)