Clinical and genetic characteristics of 15 families with hereditary hypophosphatemia: Novel Mutations in PHEX and SLC34A3

Background Hereditary hypophosphatemia is a group of rare renal phosphate wasting disorders. The diagnosis is based on clinical, radiological, and biochemical features, and may require genetic testing to be confirmed. Methodology Clinical features and mutation spectrum were investigated in patients with hereditary hypophosphatemia. Genomic DNA of 23 patients from 15 unrelated families were screened sequentially by PCR-sequencing analysis for mutations in the following genes: PHEX, FGF23, DMP1, ENPP1, CLCN5, SLC34A3 and SLC34A1. CytoScan HD Array was used to identify large deletions. Results Genetic evaluation resulted in the identification of an additional asymptomatic but intermittent hypophosphatemic subject. Mutations were detected in 21 patients and an asymptomatic sibling from 13 families (86.6%, 13/15). PHEX mutations were identified in 20 patients from 12 families. Six of them were novel mutations present in 9 patients: c.983_987dupCTACC, c.1586+2T>G, c.1206delA, c.436+1G>T, c.1217G>T, and g.22,215,887–22,395,767del (179880 bp deletion including exon 16–22 and ZNF645). Six previously reported mutations were found in 11 patients. Among 12 different PHEX mutations, 6 were de novo mutations. Patients with de novo PHEX mutations often had delayed diagnosis and significantly shorter in height than those who had inherited PHEX mutations. Novel compound heterozygous mutations in SLC34A3 were found in one patient and his asymptomatic sister: c.1335+2T>A and c.1639_1652del14. No mutation was detected in two families. Conclusions This is the largest familial study on Turkish patients with hereditary hypophosphatemia. PHEX mutations, including various novel and de novo variants, are the most common genetic defect. More attention should be paid to hypophosphatemia by clinicians since some cases remain undiagnosed both during childhood and adulthood.


Introduction
Hereditary hypophosphatemia or hypophosphatemic rickets (HR) when it occurs in children is a group of rare renal phosphate-wasting disorders with a prevalence of 3.9 per 100,000 live births [1]. It is characterized by hypophosphatemia and bone mineralization defects such as rickets and osteomalacia [2][3][4]. The patients usually present with a spectrum of clinical features such as growth retardation with disproportioned short stature, enlargement of growth plates of rapidly growing bones, lower limb deformities, bone pain, loss of teeth, and dental abscess.
PHEX and DMP1 negatively regulate the expression of FGF23, a novel phosphaturic hormone produced in osteocytes, which is secreted into the circulation after undergoing O-glycosylation by GALNT3 [25,26]. FGF23 is a key regulator of phosphate reabsorption and 1,25 (OH) 2 D 3 synthesis in the proximal renal tubules [27,28]. FGF23 levels are increased in patients with XLHR, ADHR, ARHR1, and ARHR2 leading to phosphaturia, low serum phosphate, and low serum 1,25(OH) 2 D 3 level due to its inhibition of vitamin D 1-α hydroxylase. However, in patients with HCINF2, HHRH, and XLRHR, FGF23 levels are normal or lownormal, suggesting an FGF23-independent renal tubular defect [29]. In contrast to patients with XLHR, ADHR or ARHP, who are usually treated with high doses of alphacalcidol or calcitriol and multiple daily doses of oral phosphate, oral phosphate supplementation alone is sufficient for patients with HHRH.
In our previous studies, 27 patients from 16 unrelated Turkish families were investigated [30][31][32]. In the present study, we analyzed clinical and genetic characteristics of additional patients from 15 unrelated families from a different region of Turkey.

Patients
Parents and available siblings of index patients (n = 51) from 15 unrelated Turkish families were enrolled to the study. Hereditary hypophosphatemia was diagnosed based on clinical and laboratory assessment. The clinical and laboratory data at the time of diagnosis were summarized in Table 1. The cases suspected of having renal failure, malabsorption, renal tubular acidosis and other tubulopathies that cause secondary hypophosphatemia (e.g., Fanconi's syndrome, glycogen storage disease, and galactosemia), vitamin D-dependent rickets type 1 and 2 were excluded from the study. This study was approved by the institutional review board of the Dokuz Eylul University Faculty of Medicine. Written informed consent was obtained from the children and their parents.

Genomic DNA isolation
Genomic DNA from peripheral blood leukocytes was isolated using Gentra Blood Kit (Qiagen Corp, CA).

DNA amplification and sequencing
DNA samples were first analyzed for mutations in all the coding exons and intron-exon boundaries of PHEX. If no mutations were identified, we then screened for mutations in the FGF23 followed by consecutive analysis of DMP1, ENPP1, CLCN5, SLC34A3 and SLC34A1. PCR primers and conditions were described previously [11,13,22,30,33,34]. The resulting PCR products were directly sequenced using an automated ABI PRISM 3700 sequencer (Foster City, CA) or cloned into TA vector (Invitrogen, CA). Individual clones were subsequently sequenced.

Identification of a large PHEX deletion
Copy number variation in genomic DNA was analyzed by CytoScan HD Array according to the manufacturer's procedure (Affymetrix, Santa Clara, CA). The closest undeleted probes to the boundary of deleted region (copy number loss) in the PHEX gene were used as a starting point to map the breaking point. A total of 6 walking primers were used to perform PCR until a specific DNA fragment was amplified. The PCR products were directly sequenced.

RNA isolation and RT-PCR
Total RNA from peripheral blood leukocytes was extracted as described previously [35]. Two μg of total RNA were reverse-transcribed into cDNA using Promega reverse transcription system (Promega, Madison, WI). To determine the effect of a splice site mutation at c.1586 +2T>G, RT-PCR was used to amplify PHEX transcripts using the following two primers: 5'- CTATCCAGAGTTTATAATGAA-3' (forward primer located in exon 13), and 5'-CTGTTCC CCAAAAGAAAGGCTT-3' (reverse primer located in exon 16). The PCR conditions were 95˚C for 5 minutes followed by 35 cycles of amplification (95˚C for 1 min, 54˚C for min, and 72˚C for 1 min). The resulting PCR products were analyzed by gel electrophoresis and subsequently sequenced.

In silico analysis of variants
The significance of single nucleotide variant (SNV) was predicted using the following four web-based programs: Mutation Taster (http://www.mutationtaster.org), PolyPhen-2 (http:// genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), and PROVEAN (http://provean. jcvi.org/index.php). SNV was considered to be mutation if they had: low allelic frequency (<0.01) in the normal population database; predicted to be damaging or disease-causing by at least three of four prediction programs; and present only in the patient or in strict segregation with phenotype in familial cases. Biallelic or compound heterozygous mutations were considered as disease-causing mutations. Monoallelic mutations in genes with dominant inheritance were also considered as disease-causing mutations.

Statistical analysis
Student's t test or Chi-square test (two-tailed) was used to compare two groups. A p value of 0.05 or less was considered significant.

Results
Hereditary hypophosphatemia were diagnosed in 23 patients from 15 families: 17 children (mean age at diagnosis 4.9±1.9 years, age range 1.3-8.9 years), and 6 adults (mean age at diagnosis 34.2±4.5 years, age range 28-40 years). All the children had clinical and laboratory features of rickets such as genu varum deformity, metaphyseal X-ray findings, and/or fractures. The X-ray images of metaphyseal widening and irregularity, cupping and fraying of the metaphyseal region, haziness of cortical margins and epiphysis, and enlargement of the space between the epiphysis and metaphysis were present in all HR children except for X-3 and X-4. Loss of teeth and gum abscess were observed in case IX-3. Cases III-3, VI-4, and VII-3 had undergone a surgical operation for correction of severe lower limb deformities. Bone pain was not recorded in any of the cases. All the adults were diagnosed based upon relevant clinical (bone pain, walking difficulty, muscle weakness) and laboratory findings (hypophosphatemia, mild-moderate elevated PTH, increased renal phosphate loss). Ten children (58.8%, 10/17) and five adults (83.3%, 5/6) had short stature. Although affected parents had short stature, genu varum, and/or bone fractures after minor trauma starting from childhood, none was diagnosed previously. Genetic analysis identified disease-causing gene mutations in 22 subjects (21 patients and 1 asymptomatic sibling) from 13 families: 12 different PHEX mutations in 20 patients from 12 families, and two different SLC34A3 mutations in two compound heterozygous siblings, one of whom was asymptomatic. No mutation was identified in two patients from two different families. 37]. The c.1217G>T (p.C406F) is located at the same position as our previously identified mutation c.1217G>A (p.C406Y) in a Turkish patient [30]. Six previously reported PHEX mutations were found in 11 patients from 6 families: c.1645C>T (p.R549 Ã ) [38], c.1601C>T (p.P534L) [38], c. 187+1G>T [39], c.2104C>T (p.R702 Ã ) [38], c.2239C>T (p.R747 Ã ) [40], and c.1404+1del G (direct submission to PHEX mutation database: http://www.phexdb.mcgill.ca/) ( Fig 1B and Table 2). The c.1586+2T>G, c.1206delA, c.1217G>T, c.1645C>T, c. 187+1G>T, and c.2104C>T were not present in the parents and therefore were de novo mutations, accounting for 50% of identified PHEX mutations. The effect of splice donor site mutation c.1586+2T>G on mRNA splicing was investigated by RT-PCR analysis of cDNA from patient's peripheral lymphocytes. As shown in Fig 1A, the mutation resulted in exon 14 skipping. The c.1586+1G>A was reported previously without annotation of splicing consequence    [2]. Based on our current study, the mutation is likely result in exon 14 skipping as well. Most of the mutations were splice site mutations, deletions or duplication, or nonsense mutations. These mutations are predicted to cause frameshift, resulting in either truncated protein or nonsense-mediated decay. Only two missense mutations were identified: c.1601C>T (reported) and c.1217G>T (novel). We performed in silico analysis of these two missense mutations as well as two previously reported mutations that were located in the same codon as c.1217G>T (c.1217G>A, p.C406Y and c.1216T>C, p.C406R) by four web-based programs. As shown in Table 3, all of them were predicted to be disease-causing mutations. We could not amplify by PCR the region containing PHEX exon 16-22 from affected father of index patient (V-3) from family V whereas the same region was amplified from the female patient and her mother's DNA, suggesting the possible presence of a large deletion. We next performed a copy number analysis and identified the deleted region (Fig 2A). Based on the 5' and 3' undeleted probe sequences closest to the deleted region, we performed chromosome walking to identify the breakpoint. As shown in Fig 3B and 3C, the 5' breakpoint was located in the intron 15 and 7 kb from the exon 15 of the PHEX gene (X: 22,215,887); the 3' breakpoint was 103 kb from the ZNF645 gene and 130 kb from the PHEX gene (X: 22,395,767), resulting in the deletion of 179,880 bp DNA fragment. Single TAA is present at the 5' breakpoint and immediately before the 3' breakpoint, which may be used for homologous recombination (Fig 2B and 2C).
When the patients with PHEX mutation were evaluated according to the type of inheritance (de novo or inherited), height SD scores and the frequency of short stature were significantly lower in the inherited group (Table 4). Although patients with an inherited mutation was younger at the time of diagnosis when compared to the patients with a de novo mutation, this difference was not statistically significant. The patients were successfully treated with calcitriol and phosphate.

SLC34A3 mutations
Two novel SLC34A3 compound heterozygous mutations were found in the index case from Family X (X-3) and his asymptomatic sister (X-4): a splice donor site mutation c.1335+2T>A and a 14 bp deletion c.1639_1652delCGCTCCTGGGCCTG (Fig 3 and Table 2). A heterozygous c.1335+2T>A and c.1639_1652delCGCTCCTGGGCCTG were carried by his non-consanguineous father and mother, respectively. The index case, a 5-year-old boy was first evaluated by a nephrologist (SK) due to nephrocalcinosis. Hypophosphatemia and elevated ALP were also observed in addition to hypercalciuria (0.23 mg/mg urine calcium/creatinine ratio, normal <0.20) but were not considered clinically significant initially due to lack of other rachitic findings (Table 1). He had no additional symptoms or findings consistent with rickets. Oral phosphate treatment was started at the age of 14 years following the genetic diagnosis. Consequently, and increased urine calcium/creatinine ratio (0.7 mg/mg). She did not require phosphate treatment during 2 years of follow-up. Their father (Case X-1) had recurrent nephrolithiasis but mother (Case X-2) was free of symptoms. Biochemical profiles were normal in both parents.

Families without known mutations
No mutation was detected in the patients from families XI and XIII. Copy number analysis of whole genome did not reveal any large deletions as well. Case XI-3, a 2.5-year-old boy, was admitted due to skeletal deformities of his hands and feet. His parents were not relatives. Physical examination revealed a normal height (SD score -0.4), prominent widening of wrists, and genu varum. Hypophosphatemia, increased ALP level and urine phosphate excretion, normal calcium and PTH levels, and rachitic X-ray findings were found (Table 1). Vitamin D supplement due to low 25-hydroxyvitamin D3 level did not result in improvement. Calcitriol and neutral phosphate treatment with the diagnosis of HR resulted in resolution of rickets and normalization of laboratory variables.
The other patient (XIII-3) was a 7.5-year-old boy who was evaluated due to inability to walk during the past 1 year. Physical examination was normal other than widening of wrists and genu varum deformities. Laboratory and skeletal X-ray findings were consistent with HR (Table 1). Calcitriol and neutral phosphate were started, and clinical and laboratory improvement were achieved within the first year. His treatment was gradually reduced and stopped when he was 13 years old. The most recent follow-up without treatment was made when he was 21 years old. Physical examination revealed a height of 175.5 cm (-0.19 SDS) and no skeletal abnormalities. Laboratory studies showed normal calcium (9.3 mg/dL, normal 8.8-10.5), ALP (192 IU/L, normal 93-309), and 25-hydroxyvitamin D3 (20.6 ng/mL, normal >20), but low serum phosphate (1.96 mg/dL, normal 2.7-4.5), mildly increased PTH (96 pg/mL, normal 11.1-79.9), normal TRP (91.0%, normal <85%), decreased TmP/GFR (1.78, normal 2.5-4.5), and a normal urine calcium/creatinine ratio (0.01 mg/mg creatinine, normal <0.20).

Discussion
In the present study, we investigated 23 patients from 15 unrelated families with hereditary hypophosphatemia. Disease-causing mutations were found in 21 patients and one asymptomatic  [2], Denmark [5] and US [6].
The PHEX gene is located on the X chromosome and consists of 22 short exons encoding a protein of 749 amino acids [41]. It is primarily expressed in osteoblasts and osteocytes, and plays an important role in phosphate metabolism [41]. An inactivating mutation of PHEX leads to increased serum FGF23 level and thus renal phosphate loss. Most PHEX mutations found in the current study are either splice site or insertion/deletion mutations, resulting in either truncated proteins devoid of normal function or truncated mRNA likely going through nonsense-mediated decay [42]. The delay in diagnosis of our patients with de novo PHEX mutations is likely due to lack of family history and awareness by their physicians, which results in treatment delay and more severe growth retardation. We could not detect mutation in two patients from 2 families, which may indicate possible existence of a promoter mutation in the screened genes or a novel disease-causing gene, which needs to be investigated further.
We have characterized another PHEX deletion with identification of the breakpoint (g. 22,215,887-22,395,767del). The large PHEX deletion missing exon 16-22 has been reported previously but the breakpoint has never been investigated [5,43]. It is not clear whether previously reported cases have the same breakpoint or not. Interestingly, TAA repeats are also present in the 5' breakpoint of our previously characterized 40 kb PHEX deletion [31]. Unlike the 40 kb PHEX deletion which has 13 TAA tandem repeats present before the 3' breakpoint, only one TAA repeat is present before the 3' breakpoint in the current deletion, suggest that the deletion mediated by homologous recombination may not need multiple tandem repeats. The entire ZNF645 is also deleted together with exon 16-22 of the PHEX gene. ZNF645 is a RING finger protein and function as an E3 ubiquitin-protein ligase. It may play a role in human sperm production and quality control [44].
The c.1335+2T>A and c.1639_1652del14 mutations in SLC34A3 are predicted to cause frameshift and result in loss-of-function of the gene. Given the autosomal recessive inheritance of HHRH, both patient and his sister should have the disease. The apparent phenotype-genotype discordance in the patient's sister is not clearly known. Various genetic and/or epigenetic factors may contribute to the phenotype variation. A recent meta-analysis have demonstrated that nearly 25% of individuals with homozygous or compound heterozygous SLC34A3 mutations have no rickets/osteomalacia, and 50% of individuals without renal calcifications (nephrolithiasis or nephrocalcinosis), reflecting the heterogeneity in clinical presentations caused by SLC34A3 mutations [45].
In summary, we have systemically investigated genetic defects in Turkish patients with hereditary hypophosphatemia. PHEX mutation is the most common genetic defect accounting for 84% of the disease. De novo PHEX mutations are common and often contribute to the delay in diagnosis and treatment. The early diagnosis of new-born family members may be possible by screening asymptomatic children who have family history of disease-causing mutations or the knowledge of frequent de novo PHEX mutations in the population. This will enable early treatment and a better growth outcome.