Background
Hypophosphatemic rickets (HR) is a genetic disorder in which the proximal renal tubule cannot reabsorb sufficient phosphate (Pi), leading to growth retardation, rickets, and osteomalacia [
1]. The most frequent form of hereditary HR is X-linked hypophosphatemic rickets (XLHR; MIM#307800), in which 80% of familial HR and 70% of sporadic HR are diagnosed as XLHR [
2]. XLHR is caused by loss-of-function (LOF) mutations in the phosphate regulating gene with homologies to endopeptidases on the X chromosome (
PHEX).
The XLHR inheritance pattern is X-linked dominant as both males and females present with the disease, and its prevalence is 1 in 20,000 live births [
3]. The number of
PHEX mutations reported in the Human Gene Mutation Database (HGMD) [
4] is more than 400 mutations, most of which are missense and nonsense mutations, however other types including small deletions/insertions, abnormal splicing, and gross insertions/deletions have also been reported [
1,
5].
Other less common forms of hereditary HR include autosomal dominant HR (ADHR), autosomal recessive HR 1 (ARHR1), autosomal recessive HR 2 (ARHR2), hereditary HR with hypercalciuria (HHRH), and X-linked recessive HR (XRHR) which are caused by gene mutations in fibroblast growth factor 23 (FGF23), dentin matrix acidic phosphoprotein 1 (DMP1), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), solute carrier family 34, member 3 (SLC34A3), and chloride channel 5 (CLCN5), respectively.
FGF23 plays an important role in regulating phosphate and vitamin D homeostasis. PHEX, DMP1 and ENPP1 negatively regulate the expression of FGF23, which is produced in osteocytes and secreted into the circulation [
6,
7]. The deficiencies of these genes lead to excessive secretion of FGF23. Excess FGF23 causes hypophosphatemia in two ways. The expression of phosphate co-transporters in the kidney is suppressed by FGF23, leading to decreased phosphate reabsorption. FGF23 also reduces the production of 1,25-dihydroxyvitamin D (1,25(OH)
2D3) and thus decreases the absorption of phosphate in the intestine. Other biochemical findings include elevated serum alkaline phosphatase (ALP) and normal to slightly high parathyroid hormone (PTH) levels [
8,
9].
The clinical features are similar in various forms of hypophosphatemia, although this does not imply that the symptoms are identical. Children mostly present with rickets, short stature, dental abscesses, and lower limb bone deformities. Symptoms of the disease in adults include osteomalacia, bone pain, and enthesopathy [
5]. Treatment for XLHR, ADHR or ARHR, includes oral phosphate and vitamin D supplementations while, patients with HHRH are only treated with phosphate supplementation [
10]. This conventional treatment improves rickets in children. However, a significant number of patients may not show successful results [
11] and complications such as nephrocalcinosis and hyperparathyroidism have been reported in some cases [
12]. In addition to the conventional treatments, burosumab, which was approved by the US Food and Drug Administration in 2018, is used to treat XLHR. Burosumab is a human monoclonal antibody targeting FGF23, thus, significantly improves rickets, growth, and biochemical abnormalities [
8,
12].
The rarity of HR may lead to delayed diagnosis. In addition, the recent developed treatment for this disease cannot be prescribed to the patients without defining and accurately diagnosing the disease. Therefore, correct and early diagnosis of this disease is important for optimal treatment. In the Malaysian population, there is very little information about the clinical, biochemical and, most importantly, genetic features of HR [
13,
14]. Previously, only one genetic study of HR disease had been performed in Malaysia in which Sanger sequencing for
PHEX,
FGF23 and
DMP1 genes in four Malaysian HR pediatric patients revealed two
PHEX variants [
14].
In the present study, we report clinical, radiological, laboratory, and genetic data of three pediatric patients with HR in Malaysia, for which whole exome sequencing (WES) revealed three candidate pathogenic variants. Further analysis was employed to support the pathogenicity of the variants.
Discussion
HR is a rare genetic disease with genetic heterogeneity [
34]. There has been scarce genetic confirmation of HR in Malaysia. The present study was established to determine if molecular diagnosis of three Malaysian HR patients could be achieved after screening the common genes for HR. Although clinical, radiographic, and laboratory data can help diagnose HR, genetic testing is more reliable for accurate diagnosis. All patients in this study showed typical clinical, radiographic and biochemical features of HR, with no family history of HR.
In the present study, two variants in
PHEX and one variant in
DMP1 were identified using WES in three unrelated patients with HR from Malaysia. The two
PHEX variants were de novo in the patients who were both females. This reflects previous findings of a higher rate of the
PHEX de novo mutations in females than in males [
35]. This is likely due to X-linked mutations occurring more frequently on the X chromosome in paternal germ cells [
34]. The presence of de novo variants is one of the criteria to consider when establishing the pathogenicity of a variant [
31,
36]. The confirmation of paternity testing in the two families with de novo
PHEX mutations and the index patients being affected, provide strong criteria towards classifying the pathogenicity of these variants.
The de novo nonsense mutation in
PHEX (c.871C > T, p.Arg291Ter) identified in this study has frequently been reported in HR patients from several studies of other populations including European, Chinese, and Japanese which may suggest that the nucleotides of the Arg291 residue is a mutation hot spot in
PHEX and the present study confirms that p.Arg291Ter variant is a cause of HR disease in the Malay population. The patients with the p.Arg291Ter mutation in the previous studies had a similar phenotype to patient 1 including hypophosphatemia, bone deformities and radiological signs of rickets which are typical phenotype of HR [
1,
32,
37‐
48].
The novel in-frame deletion identified in this study is located in exon 19 of
PHEX (p.Gly649_Arg651del). PHEX is a member of the M13 family of membrane-bound zinc-metallopeptidases. There is a sequence motif (
642ENXADXGG
649) in exon19 of
PHEX that is highly conserved in the members of the M13 family. A previous study in neutral endopeptidase (NEP), another member of the M13 family, has shown that this conserved motif plays an important role in zinc binding and catalysis activity [
49]. The glycine 649 residue is located in the
642ENXADXGG
649 motif, therefore, the deletion of this residue may interfere with zinc binding and catalytic activity of PHEX. A study by Kinoshita et al. [
33] also revealed a missense mutation at this position (p.Gly649ASP) in three affected patients with HR from one family. In addition, Francis et al. [
32] and Zheng et al. [
1] reported a missense mutation at Arg 651 of PHEX (Arg651Pro) in two unrelated patients with HR. Although the prediction tools did not predict this deletion as disease-causing, the previous reports may indicate the importance of glycine and arginine residues at positions 649 and 651 of PHEX
, respectively, and that these positions may be mutation hot spots.
The novel splicing variant in the
DMP1 gene (c.54 + 1G > A) was also detected with WES. Canonical eukaryotic splicing relies on four conserved nucleotides, i.e. the donor sequence GT at the 5′ end of the intron, and the acceptor sequence AG at the 3′ end [
50]. The variant c.54 + 1G > A is located within the canonical site at the 5′ end of intron 2 of the
DMP1 gene. Mutations at the canonical splice sequences usually lead to single exon skipping [
51]. The prediction of aberrant splicing and the involvement of highly conserved splice site sequences, provided evidence that the variant is likely causing HR in the patient. Of note, the same nucleotide position in
DMP1 but with different base change (c.54 + 1G > C) has been reported in a Chinese family with HR disease. Two patients of the family are homozygous and the healthy parents are carriers of the c.54 + 1G > C variant. Ni et al. showed that c.54 + 1G > C in the
DMP1 gene leads to the skipping of the whole exon 2 which then disrupts the reading frame of
DMP1 [
52]. Furthermore, the c.54 + 1G > C variant has been reported in gnomAD in the heterozygous state with a minor allele frequency (MAF) < 0.1% (0.00039%).
Although this research presents novel findings from a unique ethnicity which is rarely studied, it also has several limitations. Small sample size is one of the limitations which is due to the rareness of HR disorder and small area of data collection. Therefore, statistical analysis regarding phenotype and genotype of patients could not be performed. Moreover, genetic analysis was carried out only for parent-child trios and multi-generational genetic investigation was not available. This is particularly important for the family in which the parents of an affected child were carriers of the variant. Another limitation is the absence of functional analysis to fully understand the impact of novel variants.
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