Heterozygous mutation of SLC34A1 in patients with hypophosphatemic kidney stones and osteoporosis: a case report.

Introduction

Inorganic phosphates are involved in a wide range of cellular processes, including intracellular signal transduction, energy exchange, pH buffering, bone mineralization, cellular phospholipid structure, and nucleic acid synthesis.[1] The absorption of phosphate is mainly dependent on sodium phosphate cotransporters, of which NaPi-IIa, encoded by SLC34A1, is the most important. Mutations of SLC34A1 lead to NaPi-IIa dysfunction, resulting in phosphate absorption problems and clinical symptoms associated with phosphate deficiency. Clinically, however, hypophosphatemia is rare, and patients with SLC34A1 mutations often show skeletal abnormalities such as rickets and osteomalacia. Other clinical manifestations include slow growth, a short stature, bone pain, and limb malformation.[2] Hypophosphatemia is difficult to diagnose and is easily misdiagnosed as osteoporosis, bone tumors, myopathy, and other diseases.

Current treatment of hypophosphatemia mainly involves the supplementation of neutral phosphate. This can be achieved by two methods: oral phosphorus tablets or the dissolving of compound disodium hydrogen phosphate and potassium dihydrogen phosphate in water. Both drugs have been shown to improve hypophosphatemia.[3]

Here, we present a case of a 38-year-old Chinese woman with pain in multiple bones throughout her body and difficulty with walking for more than 2 years. We obtained her clinical records and carried out genetic analysis of the proband and her family, resulting in a diagnosis of hypophosphatemic kidney stones with osteoporosis. We also explore the pathogenesis and treatment of the disease.

Materials and methods

Ethical approval

The proband and her parents provided their informed consent for participation of the study. This study was approved by the Ethics Committee of the First Hospital of Lanzhou University.

Blood collection and DNA sequencing

A total of 2 mL fasting blood was collected from the proband and her parents between 08:00 and 10:00 hours into ethylenediaminetetraacetic acid-treated tubes. Genomic DNA was extracted using the Blood Genome Column Medium Extraction Kit (Kangweishiji, Beijing, China) according to the manufacturer’s instructions. For whole exome library construction, protein-coding exome enrichment was performed using xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Inc., Coralville, IA, USA). This consists of 429,826 individually synthesized and quality-controlled probes, targeting a 39-Mb protein-coding region (19,396 genes) of the human genome and covering 51 Mb of end-to-end tiled probe space. High-throughput sequencing was performed by an Illumina NovaSeq 6000 series sequencer (PE150; Illumina, San Diego, CA, USA), and not less than 99% of the target sequence was sequenced.

Bioinformatics analysis

Raw data were processed by fastp for adapter removal and low-quality read filtering. Variant calling of paired-end reads was performed using the Burrows–Wheeler alignment tool of the Ensembl GRCh37/hg19 reference genome. Base quality score recalibration together with single nucleotide polymorphism (SNP) and short indel calling were conducted using The Genome Analysis Toolkit. SNPs and indels were screened according to the sequence depth and variant quality to obtain high-quality and reliable variants. An online system independently developed by Chigene (www.chigene.org) was used to annotate database-based minor allele frequencies (MAFs), and to determine American College of Medical Genetics (ACMG) practice guideline-based pathogenicity of every yielded gene variant. The system also provided a serial software package for conservative analysis and protein product structure prediction. Databases for MAF annotation included 1,000 Genomes, dbSNP, ESP, ExAC, and the Chigene in-house MAF database. PROVEAN, SIFT, Polypen2_hdiv, Polypen2_hvar, MutationTaster, M-Cap, and REVEL software packages (database dbNSFP4.0b1a) were used to predict protein product structure variation for the prediction of pathogenicity. As a prioritized pathogenicity annotation to ACMG guidelines, OMIM, HGMD, and ClinVar databases were used as conferences of pathogenicity of every variant. To predict functional changes of variants on splicing sites, MaxEntScan, dbscSNV, and GTAG software packages were used instead of product structure prediction software.

Results

Clinical analysis

Two years previously, the patient presented with weakness and pain in her lower limbs, which became progressively aggravated. She has no history of chronic disease such as renal insufficiency, and no long-term use of glucocorticoids or other medication history. Physical examination revealed pain in the bilateral ribs and the muscle strength of both lower limbs was reduced. Her mother suffered a femoral neck fracture from a fall at the age of 45. At the age of 58, her femoral head has now become necrotic so she has undergone surgery.

Biochemical analysis

Biochemical tests of the patient revealed significantly reduced levels of blood phosphorus (AU5800 Clinical Chemistry Analyzer, Beckman Coulter Inc., Brea, CA, USA) ranging from 0.27 to 0.9 mmol/L (normal range, 0.97–1.6 mmol/L), and urinary phosphorus ranged from 9.86 to 19.3 mmol/24 hours (normal range, 9.7–42.0 mmol/24 hours). However, blood and urinary calcium were normal, and immunoreactive parathyroid hormone levels ranged from 76 to 237 pg/ml (normal range, 11–81 pg/ml). Routine urine tests were also normal. The ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ranged from 1.48 to 1.92 mg/dl, indicating a renal phosphate leak (related data are shown in Table 1). Biochemical examination of the proband’s mother revealed low serum phosphate, ranging from 0.38 to 0.79 mmol/L. Urinary phosphorus ranged from 8.7 to 18.3 mmol/24 hours, and blood and urinary calcium levels were normal.

Table 1.

The proband’s biochemical parameters.

Date	08/23/2016	10/18/2016	09/21/2017	01/16/2018	11/07/2018	11/01/2019
Blood phosphorus (mmol/L）	0.5	0.42	0.3	0.32	0.27	0.9
24 hour urinary phosphorus (mmol/24 hours）	–	11.1	–	9.86	19.3	–
Blood calcium (mmol/L）	2.38	2.30	2.42	2.24	2.33	2.36
24 hour urinary calcium (mmol/24 hours)	–	4.6	–	5.33	7.2	–
iPTH (pg/ml）	76	101.5	101	128.9	237	–

iPTH, immunoreactive parathyroid hormone.

Color ultrasound (KR-8288V, Kaier Medical Appliance Co., Ltd., Jiangsu, China) indicated stones in the left kidney of the proband and in the left kidney of her mother. A whole body scan using emission computed tomography (BrightView SPECT; Philips Healthcare, Best, the Netherlands) showed abnormally active bone mineral metabolism in knee, hip, shoulder, and elbow joint areas of the patient, leading to a diagnosis of systemic metabolic bone disease (Figure 1a).

Figure 1.

(a) Proband mutation site (c.1753 T>C) in exon 13 of SLC34A1. (b) Mutation sites in the mother of the proband (c.1753 T>C). (c) Sequencing of the father of the proband showing wild-type sequence (c.1753 T).

Digital radiography using the Axiom Aristos VX DR (Siemens Healthcare, Erlangen, Germany) exposed cervical bone and lumbar vertebrae hyperplasia, and osteoporosis in the hands and feet of the patient (Figure 1b, 1c). Magnetic resonance imaging using a Magnetom Amira MRI scanner (Siemens Healthcare) revealed a decreased bone signal in the lumbar spine. Dual-energy x-ray absorption with the Lunar iDXA (GE Healthcare, Piscataway, NJ, USA) confirmed osteoporosis in a bone density examination of both the proband (Table 2) and her mother.

Table 2.

Bone density analysis of the proband.

Section	Bone mineral density (g/cm2)	Adults		Compared with healthy individuals of the same age
		(%)	T-value	(%)	Z- value
L1	.728	71	–2.5	76	–2.0
L2	.784	71	–2.7	76	–2.1
L3	.803	70	–2.9	74	–2.3
L4	.773	68	–3.1	72	–2.5
L1-4	.774	69	–2.8	74	–2.3

DNA sequencing

Whole exon sequencing identified three potentially pathogenic genes: SLC34A1, HRAS, and TRH. TRH mutations induce thyrotropin-releasing hormone deficiency syndrome,[4] which is not related to hypophosphatemia, while HRAS mutations cause linear sebaceous nevus syndrome,[5] which is inconsistent with the clinical manifestations of the proband. Therefore, these two genes were excluded as pathogenic. Hypophosphatemic kidney stones with osteoporosis caused by SLC34A1 mutations were consistent with the clinical features of the proband, so mutated SLC34A1 was considered causative of disease.

Sequencing identified a mutation in the 13th exon of SLC34A1 in the proband: c.1753 T>C. This substitution of a T with a C at position 1753 of SLC34A1 (Figure 2a) resulted in the replacement of serine by proline at position 585 of the protein (p. S585P). This is a novel mutation that has not been previously reported in the ExAC browser. The proband’s mother was also shown to harbor the same mutation (Figure 2b), but the proband’s father has the wild-type sequence (Figure 2c).

Figure 2.

The mutated amino acid at position 585 (red arrow) is located inside the structural domain of the Phosphate:Na+ Symporter Family of proteins according to NCBI/Structure/Cdd.

Computational mutation prediction of the SLC34A1 conserved domain using the NCBI Conserved Domain Database (Cdd) revealed that the mutated amino acid at position 585 is located inside the structural domain of the Phosphate:Na+ Symporter family of transport and binding proteins (Figure 3). Sequence-based mutation prediction for p. S585P was probably damaging according to NCBI/Structure/Cdd (ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The mutation was predicted to result in a decrease in phosphate uptake compared with wild-type SLC34A1.

Figure 3.

(a) Whole body bone scan emission computed tomography of the patient. (b) Lumbar spine digital radiography of the patient. (c) Pelvis digital radiography of the patient.

Based on the above findings, and combined with the patient’s clinical manifestations, she was diagnosed with hypophosphatemic kidney stones with osteoporosis. She was administered 4 mL of 253 g of disodium hydrogen phosphate and 126 g of potassium dihydrogen phosphate dissolved in 1 L water every 5 hours, and calcitriol 0.5 µg/day. After 1 month, her bone pain was relieved, and she was able to walk slowly and independently after more than 2 months. Her blood calcium had normalized after 1 month, and her blood phosphorus level increased to 0.9 mmol/L after 2 months and normalized after 4 months.

Discussion

Inorganic phosphate plays an important role in bioenergetics, metabolic regulation, intracellular signal transduction, cell proliferation, and the formation of bone and cell membrane structures in vivo. Approximately 85% of systemic phosphate accumulates in bones and teeth, 14% in soft tissue, and 1% as free phosphate in extracellular fluid. Phosphorus reabsorbed protein–sodium phosphate cotransporter is divided into three types: NaPi-I, NaPi-II, NaPi-III, encoded by SLC17, SLC34, and SLC20, respectively.[6] Kidney mRNA contents were previously reported to be 15%, 84%, and 1%, respectively, of which NaPi-II was the most important.[7,8] NaPi-II has three subtypes, NaPi‐IIa, NaPi‐IIb, and NaPi‐IIc which are encoded by SLC34A1, SLC34A2, and SLC34A3, respectively.[9] NaPi‐IIa and NaPi‐IIc are localized specifically in the apical brush border membrane of renal proximal tubule cells, whereas NaPi‐IIb is found in many tissues, including the luminal brush border membrane of the small intestine and alveolar type II epithelial cells.[10,11] Studies of knockout mice and human disease have shown the importance of these transporters at the whole body level. Mice lacking NaPi‐IIa exhibit hypophosphatemia and hyperphosphaturia,[12] while those lacking NaPi‐IIb die in utero.[13] Human NaPi‐IIc mutations cause hereditary hypophosphatemic rickets with hypercalciuria.[14]

SLC34A1 is located on chromosome 5q35 and consists of 13 exons and 12 introns.[11,15] NaPi-IIa consists of 639 amino acids and is mainly expressed on the apical brush border membrane of the proximal tubule of the kidney. SLC34A1 mutations affect the function of NaPi-IIa, which in turn affects the reabsorption of phosphorus. This indicates that SLC34A1 is an important determinant of plasma phosphate levels and that it regulates human renal phosphate homeostasis. SLC34A1 mutations can cause a range of clinical phenotypes, including infant hypercalcemia, proximal renal Fanconi syndrome, and hypophosphatemic kidney stones, all of which are associated with low phosphorus levels. We excluded the first two diagnoses according to the patient’s clinical characteristics and test data, so she was ultimately diagnosed with hypophosphatemic kidney stones with osteoporosis.

Our genetic analysis identified the novel mutation c.1753 T>C in exon 13 of SLC34A1 in the proband and her mother, which results in substitution of serine by proline at position 585 of NaPi-IIa. The mutation was found to be inherited in an autosomal dominant manner. Previous studies showed that cartilage and ligament tissues in the teeth and bones become weakened when the ratio of proline and hydroxyproline is unbalanced, which could be the cause of the clinical manifestations such as bone pain in the proband and her mother. Based on these findings, the proband was diagnosed with hypophosphatemic kidney stones with osteoporosis which was alleviated after the administration of disodium hydrogen phosphate, potassium dihydrogen phosphate, and calcitriol.

In conclusion, hypophosphatemic kidney stones and osteoporosis can be diagnosed by genetic analysis, which would reduce the rate of misdiagnosis. Thus, in cases of systemic bone pain, fractures, and other symptoms and when the examination suggests hypophosphatemia and SLC34A1 mutation, hypophosphatemic kidney stones and osteoporosis should be considered when there is no notable improvement in symptoms following calcium supplementation.