Phenotypic characterization of X-linked hypophosphatemia in pediatric Spanish population

Background X-linked hypophosphatemia (XLH) is a hereditary rare disease caused by loss-of-function mutations in PHEX gene leading tohypophosphatemia and high renal loss of phosphate. Rickets and growth retardation are the major manifestations of XLH in children, but there is a broad phenotypic variability. Few publications have reported large series of patients. Current data on the clinical spectrum of the disease, the correlation with the underlying gene mutations, and the long-term outcome of patients on conventional treatment are needed, particularly because of the recent availability of new specific medications to treat XLH. Results The RenalTube database was used to retrospectively analyze 48 Spanish patients (15 men) from 39 different families, ranging from 3 months to 8 years and 2 months of age at the time of diagnosis (median age of 2.0 years), and with XLH confirmed by genetic analysis. Bone deformities, radiological signs of active rickets and growth retardation were the most common findings at diagnosis. Mean (± SEM) height was − 1.89 ± 0.19 SDS and 55% (22/40) of patients had height SDS below—2. All cases had hypophosphatemia, serum phosphate being − 2.81 ± 0.11 SDS. Clinical manifestations and severity of the disease were similar in both genders. No genotype—phenotype correlation was found. Conventional treatment did not attenuate growth retardation after a median follow up of 7.42 years (IQR = 11.26; n = 26 patients) and failed to normalize serum concentrations of phosphate. Eleven patients had mild hyperparathyroidism and 8 patients nephrocalcinosis. Conclusions This study shows that growth retardation and rickets were the most prevalent clinical manifestations at diagnosis in a large series of Spanish pediatric patients with XLH confirmed by mutations in the PHEX gene. Traditional treatment with phosphate and vitamin D supplements did not improve height or corrected hypophosphatemia and was associated with a risk of hyperparathyroidism and nephrocalcinosis. The severity of the disease was similar in males and females.

We here report the clinical manifestations at diagnosis and follow-up of a large series of Spanish patients included in the online database RenalTube [18]. This study is justified at least by the following reasons: (1) XLH is a rare disease and few publications provide data on large series of patients; (2) XLH has a broad phenotypic variability and additional information is required to better characterize the clinical spectrum of the disease and to explain why the number of cases diagnosed usually does not correspond to the estimated prevalence of the disease; (3) It is important to share data of patients with genetically confirmed XLH in order to facilitate the finding of a potential phenotype-genotype correlation and to have current data that can be compared for the assessment of the new therapies.

Results
Forty-eight patients included in the RenalTube database with the diagnosis of XLH confirmed by defect-of-function variants found in the PHEX gene were analyzed. All variants had been identified as pathogenic. Sixteen patients (33%) had variants with strong evidence of pathogenicity (nonsense, frameshift, deletions) while the other 32 (67%) harbored variants with very strong evidence of pathogenicity (SNPs). Demographic and genetic data from patients are shown in Table 1. Patients were from 39 families and were being followed in pediatric nephrology units of 17 Spanish hospitals (Fig. 1). Fifteen patients were males and 33 females. Median age at diagnosis was 2.0 (IQR 2.6) years and the age ranged from 3 months to 8 years 2 months.
Presenting manifestations are shown in Table 2 for each patient. Bone deformities and radiological signs of active rickets were the most frequent findings leading to diagnosis. Ten patients were diagnosed because of family screening. Age at diagnosis of these patients was no different from that of the rest of the series as it ranged from 0.5 to 8 years with a median age of 1.04 years.
No differences were found between males and females for clinical manifestations, growth impairment or biochemical data at diagnosis. Likewise, no genotype-phenotype correlation was found. Actually, even patients within the same family presented different severity of clinical and biochemical manifestations.
Eight out of 24 patients with renal ultrasounds at last follow-up presented nephrocalcinosis (Table 5).

Discussion
This study provides a current description of the phenotypic characteristics of a large cohort of Caucasian pediatric patients with XLH genetically confirmed. The sample is a broad representation of the Spanish children with XLH, coming from several hospitals scattered through the country and provides data at diagnosis and after a median follow-up of 7.42 years. The study confirms that growth retardation, bone deformities and active lesions of rickets are the main presenting manifestations of the disease, within a wide spectrum of symptoms. No significant differences were found between males and females as for the severity of the disease. It is of interest that a broad spectrum of PHEX gene variants, all of them already described as pathogenic, was found and no mutation was specifically prevalent in Spanish population. All variants were classified as pathogenic according to the American College of Medical Genetics and Genomics consensus [19]. There was a high phenotypical variability even among family members harboring the same mutations, suggesting that other genes and environmental factors may affect the severity of XLH, as reported by other authors [20,21].
In addition, the study shows that conventional treatment with phosphate supplements and vitamin D metabolites does not lead to persistent correction of hypophosphatemia or reduction of renal wasting of phosphate and does not modify the circulating levels of calcitriol. Unfortunately, this study does not provide information on circulating FGF23 levels, given its retrospective design. At the last follow-up visit, 11 out of 25 patients had serum PTH values mildly elevated. In XLH, development of hyperparathyroidism is thought to be related with the pharmacological administration of phosphate [22]. Eight patients developed nephrocalcinosis during the follow-up period, a finding linked to the administration of phosphate and vitamin D that usually does not result in subsequent clinical complications [16].
Though conventional treatment has been described to heal active signs of rickets and may improve bone deformities [23], this study confirms that it does not lead to catch-up growth. Mean height Z score of the group of patients remained low, − 1.89 at diagnosis versus − 1.94 at the last visit, although Fig. 3 indicates that the individual patients' response varied from marked improvement to worsening of growth impairment. Two patients, VII. 3   had BMI greater than + 1.00 SDS at the last follow-up visit. This percentage of 25% corresponds to the normal distribution of reference population and it indicates that tendency to overweight and obesity was not found in the group of XLH patients here reported, unlike other series that have recently drawn attention to these complications likely related with the sedentary life and restricted mobility of these patients [11]. In this regard, a slight but significant increase in weight was found during the follow-up period in our series. Our study presents methodological limitations inherent to the retrospective analysis and to the fact that patients' information was extracted from a database in which some data were missing and cannot be recovered. It is also of note the lack of information on the degree of adherence to medication of each patient as well as the different monitoring protocols among the participating centers. However, it is an observational clinical study describing a large cohort of Spanish pediatric patients with genetically confirmed XLH and it provides current and interesting information on the clinical and biochemical features of the disease, at diagnosis and follow-up after conventional treatment. Our findings could be used as reference for further studies using burosumab treatment.

Conclusions
This study confirms that growth retardation and rickets were the most prevalent clinical manifestations at diagnosis in a large series of Spanish pediatric patients with XLH confirmed by identification of pathogenic variants in the PHEX gene. Traditional treatment with phosphate supplements and calcitriol did not improve height or corrected hypophosphatemia and was associated with a risk of hyperparathyroidism and nephrocalcinosis. The severity of the disease was similar in males and females and no phenotype-genotype association was found.

Patients and methods
The RenalTube database including 48 patients, 15 males and 33 females, with the diagnosis of XLH confirmed by defect-of-function mutations found in the PHEX gene was retrospectively reviewed to obtain demographic information and clinical and biochemical manifestations at diagnosis and at the last annual follow-up. Genetic information was confirmed and formatted according to       [27]. All patients received treatment with phosphate supplementation (dose range of phosphorus element: 27-90 mg/kg/day at last follow-up) and vitamin D metabolites (dose range: 0.25-1.5 µg/day at last followup), according to the criteria and indications given by their physicians (Table 5). Two patients (VII.3 and XV.1) received growth hormone treatment. None of them received burosumab treatment.
Information in RenalTube database was downloaded and formatted to an Excel database. All fields but reasons for consultation and genetic information were multichoice or numeric format.
Chi squared test was used to analyze differences between sex for binary (Yes/No) fields (growth retardation, bone deformities). F-test was used to assess variance equality between sex for anthropometric and biochemical values. T-test was used to analyze differences between sex for anthropometric and biochemical values. Paired T-test for means was used to analyze differences between diagnosis and last follow-up for anthropometric and biochemical values. T-test for unequal variances was used to compare age at diagnosis for patients with and without family history of the disease.
Phenotype-genotype correlation was assessed by isolating the most severe phenotypes (lowest serum concentrations, most severe growth retardation, highest levels of alkaline phosphatases) and comparing genetic mutations in these patients looking for big deletions, SNPs with entirely different amino acids or nonsense mutations.