An update on clinical presentation and responses to therapy of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH)

Abstract

Pathogenic variants in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Here, we report a pooled analysis of clinical and laboratory records of 304 individuals from 145 kindreds, including 20 previously unreported HHRH kindreds, in which two novel SLC34A3 pathogenic variants were identified. Compound heterozygous/homozygous carriers show above 90% penetrance for kidney and bone phenotypes. The biochemical phenotype for heterozygous carriers is intermediate with decreased serum phosphate, tubular reabsorption of phosphate (TRP (%)), fibroblast growth factor 23, and intact parathyroid hormone, but increased serum 1,25-dihydroxy vitamin D, and urine calcium excretion causing idiopathic hypercalciuria in 38%, with bone phenotypes still observed in 23% of patients. Oral phosphate supplementation is the current standard of care, which typically normalizes serum phosphate. However, although in more than half of individuals this therapy achieves correction of hypophosphatemia it fails to resolve the other outcomes. The American College of Medical Genetics and Genomics score correlated with functional analysis of frequent SLC34A3 pathogenic variants in vitro and baseline disease severity. The number of mutant alleles and baseline TRP (%) were identified as predictors for kidney and bone phenotypes, baseline TRP (%) furthermore predicted response to therapy. Certain SLC34A3/NPT2c pathogenic variants can be identified with partial responses to therapy, whereas with some overlap, others present only with kidney phenotypes and a third group present only with bone phenotypes. Thus, our report highlights important novel clinical aspects of HHRH and heterozygous carriers, raises awareness to this rare group of disorders and can be a foundation for future studies urgently needed to guide therapy of HHRH.

Keywords: HHRH; SLC34A3; phosphate therapy; renal calcifications.

Figure 1.. Frequency of renal and bone phenotypes is increased in carriers of SLC34A3 pathogenic variants.

Frequency of renal and bone phenotypes in compound heterozygous/homozygous carriers (comp/hom, n=90; a), heterozygous carriers (het, n=141; b), and wild type relatives (wt, n=29; c). Individuals without data on bone or renal phenotype (symptomatic/asymptomatic) were excluded (n=44). Frequency of renal and bone phenotypes in comp/hom carriers (n=60, d), het carriers (n=37; e), and wt relatives (n=6; f) with age < 18 y. Frequency of renal and bone phenotypes in adult comp/hom carriers (N=28, g), het carriers (n=93; h), and wt relatives (n=18; i). Individuals without age data were excluded in d-h (n=18). ^&P < 0.05 vs. het; ^#P < 0.05 vs. wt; ^$P < 0.05 age < 18 y. vs. adult. For individual renal and bone phenotypes see Table 1 and Supplementary Figure S2c-d.

Figure 2.. Summary statistics of carriers of SLC34A3 pathogenic variants among compound heterozygous/homozygous carriers (comp/hom), heterozygous carriers (het), and wild type (wt) relatives.

SLC34A3 pathogenic variant carriers show decreased serum phosphate (a), TmP/GFR (b), TRP (%; c), total hip BMD (f), serum iPTH (g), and serum FGF23 (i); increased serum 1,25(OH)₂D (d), urine calcium excretion (e), and serum ALP (h). ALP, alkaline phosphatase. FGF23, fibroblast growth factor 23. iPTH, intact parathyroid hormone. TmP/GFR, tubular maximum phosphate reabsorption per glomerular filtration rate. TRP, tubular reabsorption of phosphate. U-Ca/Crea, urinary calcium/creatinine ratio. %UL, % of upper limit. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns=not significant.

Figure 3.. Variable response to oral phosphate supplementation.

Comparison of pre-treatment and post-treatment parameters, including serum phosphate (a), serum iPTH (b), urine calcium excretion (c), L1-L4 BMD z-score (d), ALP (e), serum 25(OH)D (f), serum 1,25(OH)₂D (g), TmP/GFR (h), and serum calcium(i). Comp/hom-pre, pre-treatment levels of compound heterozygous/homozygous (comp/hom) carriers; comp/hom-post, post-treatment levels of comp/hom carriers; het-pre, pre-treatment levels of heterozygous (het) carriers, het-post, post-treatment levels of het carriers. %UL, % of upper limit. For other abbreviations see legend Figure 2.

Figure 4:. ACMG score is negatively associated with the Na-dependent phosphate uptake by X. oocytes expressing wild type (wt) and mutant SLC34A3/NPT2c.

(A) X. oocytes were injected with 25 ng capped RNA/oocyte, encoding for wt or nine missense pathogenic variants of human SLC34A3/NPT2c. ³³P-uptake was determined (mean +/− SEM performed with 5 oocytes per point in triplicate); *P < 0.05 vs. wt. (B) The correlation of ACMG score to the ratio of absolute phosphate uptake (% of wt). Note: there is an overlay at ACMG score 4 for three variants (x-axis=4; y-axis=0) and at ACMG score 5 for two variants (x-axis=5; y-axis=0). r, Pearson correlation coefficient.

Figure 5.. ACMG score is associated with the phenotype.

SLC34A3 pathogenic variant carriers with renal and/or bone phenotype have a higher ACMG score (a-c). Comparisons of ACMG scores after grouping parameters into four quartiles (Q1-Q4), serum phosphate z-score (d), TmP/GFR z-score (e), iPTH (f), 1,25(OH)₂D (g), urine calcium excretion (h) or ALP z-score (i). %UL, % of upper limit. For other abbreviations see legend Figure 2.

Figure 6.. Response to therapy correlates with baseline TRP (%).

The association of baseline TRP (%) with delta serum phosphate (a), iPTH (b), 25(OH)D (c), and 1,25(OH)₂D (d). delta value = post-treatment value – pre-treatment value. %UL, % of upper limit. r, Pearson correlation coefficient.

Figure 7.

Predicted model of SLC34A3/NPT2c shows scaffold domains in purple and transport domains in orange and localization of pathogenic variants identified in individuals with HHRH or IH. (a) Arrows mark insertion, deletion or intronic position of pathogenic variants. *pathogenic variants found to have incomplete responses to therapy with oral phosphate supplementation (see also Supplementary Figure S4). The red box marks the pathogenic variant presenting bone phenotype only, and the blue box marks the pathogenic variant presenting renal phenotype only. (b) Predicted three-dimensional structure of SLC34A3/NPT2c (https://alphafold.ebi.ac.uk/entry/Q8N130) shows the location of missense pathogenic variants (n=7) found to be have incomplete responses to therapy. These specific seven amino acids are illustrated as a stick model, with carbon atoms represented in green, oxygen atoms in red, and nitrogen atoms in blue sticks. Panel b was built using PyMOL molecular visualization system (https://pymol.org/2/).