Biol. Chem. 2015; 396(1): 27–33
Lamei Yuan, Song Wu, Hongbo Xu, Jingjing Xiao, Zhijian Yang, Hong Xia, An Liu, Pengzhi Hu, Anjie Lu, Yulan Chen, Fengping Xu and Hao Deng*
Identification of a novel PHEX mutation in a Chinese family with X-linked hypophosphatemic rickets using exome sequencing Abstract: Familial hypophosphatemic rickets (HR), the most common inherited form of rickets, is a group of inherited renal phosphate wasting disorders characterized by growth retardation, rickets with bone deformities, osteomalacia, poor dental development, and hypophosphatemia. The purpose of this study was to identify the genetic defect responsible for familial HR in a four-generation Chinese Han pedigree by exome sequencing and Sanger sequencing. Clinical features include skeletal deformities, teeth abnormalities, hearing impairments and variable serum phosphate level in patients of this family. A novel deletion mutation, c.1553delT (p.F518Sfs*4), was identified in the X-linked phosphate regulating endopeptidase homolog gene (PHEX). The mutation is predicted to result in prematurely truncated and loss-of-function PHEX protein. Our data suggest that exome sequencing is a powerful tool to discover mutation(s) in HR, a disorder with genetic and clinical heterogeneity. The findings may also provide new insights into the cause and diagnosis of HR, and have implications for genetic counseling and clinical management. Keywords: exome sequencing; frameshift; genetic counseling; hypophosphatemic rickets; PHEX gene.
*Corresponding author: Hao Deng, Center for Experimental Medicine, the Third Xiangya Hospital, Central South University, Changsha 410013, China; and Department of Neurology, the Third Xiangya Hospital, Central South University, Changsha 410013, China, e-mail: [email protected] Lamei Yuan, Hongbo Xu, Zhijian Yang and Hong Xia: Center for Experimental Medicine, the Third Xiangya Hospital, Central South University, Changsha 410013, China Song Wu and Anjie Lu: Department of Orthopedics, the Third Xiangya Hospital, Central South University, Changsha 410013, China Jingjing Xiao, Yulan Chen and Fengping Xu: BGI-Shenzhen, Shenzhen 518083, China An Liu: Department of Otolaryngology-Head Neck Surgery, the Third Xiangya Hospital, Central South University, Changsha 410013, China Pengzhi Hu: Department of Radiology, the Third Xiangya Hospital, Central South University, Changsha 410013, China
DOI 10.1515/hsz-2014-0187 Received April 17, 2014; accepted July 20, 2014; previously published online July 23, 2014
Introduction Familial hypophosphatemic rickets (HR) is the most common inherited form of rickets. It was first described by Albright et al. in 1937 (Goji et al., 2006; Baroncelli et al., 2012). Familial HR consists of a group of renal phosphate wasting disorders with hypophosphatemia and inappropriately low or normal serum 1,25-dihydroxyvitamin D (1,25(OH)2D) levels (Morey et al., 2011). Familial HR is characterized by marked clinical variability of growth retardation, childhood rickets, osteomalacia, and poor dental development (Dixon et al., 1998; Goji et al., 2006; Morey et al., 2011; Santos et al., 2013). In addition, extraskeletal bone formation, osteoarthritis, limitation of joint mobility, and occasionally spinal cord compression may occur (Dixon et al., 1998). It was reported that average incidence of HR is 3.9 per 100,000 in children between age 0 and 0.9 years, and a prevalence of 4.8 per 100,000 in those younger than 15 years (Beck-Nielsen et al., 2009). Six types of hereditary HR have been identified, including the common X-linked dominant HR (XLHR, MIM 307800), the occasional autosomal dominant HR (ADHR, MIM 193100), autosomal recessive HR (ARHR1, MIM 241520, and ARHR2, MIM 613312), the rare X-linked recessive HR (MIM 300554), and two peculiar rare forms of HR associated with hypercalciuria (HHRH, MIM 241530) and hyperparathyroidism (MIM 612089) (Morey et al., 2011; Baroncelli et al., 2012; Santos et al., 2013). HR may be caused by mutations of genes involved in the complex regulation of phosphate metabolism (Baroncelli et al., 2012). To date, at least seven disease loci and six disease-causing genes, including the X-linked phosphate regulating endopeptidase homolog gene (PHEX, MIM 300550), the fibroblast growth factor 23 gene (FGF23, MIM 605380), the dentin matrix acidic phosphoprotein 1 gene (DMP1, MIM 600980), the ectonucleotide pyrophosphatase/phosphodiesterase 1 gene (ENPP1,
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28 L. Yuan et al.: Identification of a new PHEX mutation in XLHR MIM 173335), the chloride channel voltage-sensitive 5 gene (CLCN5, MIM 300008) and the solute carrier family 34 (sodium/phosphate cotransporter) member 3 gene (SLC34A3, MIM 609826), have been reported (Baroncelli et al., 2012). Among them, XLHR is the most common form, and it is caused by inactivating mutations in the PHEX gene, which is associated with increased circulating FGF23 levels (Baroncelli et al., 2012; Santos et al., 2013). XLHR patients exhibit marked phenotypic variability, and some may have normal growth (Makras et al., 2008; Baroncelli et al., 2012). The main purpose of this study was to identify the gene responsible for familial HR in a four-generation Chinese pedigree. A novel frameshift mutation (c.1553delT, p.F518Sfs*4) in the PHEX gene, disrupting the overall integrity of the PHEX protein, was found to co-segregate with HR patients in this family and be absent in normal controls, indicating that it is a pathogenic mutation.
Results Clinical findings Detailed clinical, laboratory, auditory and radiological characteristics are summarized in Table 1. All patients
in the family had short stature and apparent lower extremity deformities. Blood biochemical tests revealed various degrees of low serum phosphate, normal serum calcium, elevated parathyoid hormone (PTH) and alkaline phosphatase (ALP) levels. Three patients complained of gait disturbance, bone pain and fatigue. A patient (II:1, Figure 1) lost all the teeth in her thirties, and her daughter (III:1) also complained about losing teeth in early adulthood. The 13-year-old proband (IV:1) had nearly normal tooth development, but suffered from hearing impairment. External auditory meatus and tympanic membrane of all patients were normal. Two patients (II:1 and IV:1) showed hearing impairments. One (II:1) was 62 years old, hence presbyacusis cannot be ruled out (Fishman et al., 2004). The other was a young female (IV:1) with bilateral mixed hearing loss. The severity of some clinical features, including bone pain, dental anomalies and hearing impairment varied among patients of this family. The proband was diagnosed with HR based on diagnostic criteria. According to the medical records, there were an additional three patients (II:1, III:1 and IV:2) who were also diagnosed with HR manifested disabilities in walking and severely growth retardation, and positive family history confirmed the diagnosis of familial HR in this family (Morey et al., 2011; Kang et al., 2012).
Table 1 Clinical, laboratory, auditory, radiological and genetic data of four patients with the PHEX c.1553delT mutation. Subject
II:1
Gender Age (years) Genotype Weight (kg) Height (cm) Growth retardation Bowing of legs Disabilities in walking Bone pain Skeletal deformities Dental anomalies Hearing impairment
Serum phosphate Serum calcium Serum ALP Serum PTH Urine phosphate Urine calcium Radiological changes
III:1
Female 62 Heterozygote 46 122 Yes Yes Yes Yes Yes Premature tooth loss, edentulous jaw Sensorineural hearing loss (R), mixed hearing loss (L) ↓ N ↑ ↑ ↑ ↓ Yes
IV:1
Female 33 Heterozygote 52 117 Yes Yes Yes Yes Yes Premature tooth loss, periodontitis No ↓ N ↑ ↑ ↑ ↓ Yes
IV:2
Female 13 Heterozygote 27 116 Yes Yes Yes Yes Yes Enamel hypoplasia Bilateral mixed hearing loss ↓ N ↑ ↑ ↑ ↓ Yes
Male 6 Hemizygote 17 68 Yes Yes Yes No Yes Hypodontia, enamel hypoplasia, prematureloss deciduous tooth, dental caries N/A ↓ N ↑ ↑ ↑ ↓ Yes
ALP, alkaline phosphatase; L, left; N, normal values; N/A, not available; PHEX, the X-linked phosphate regulating endopeptidase homolog gene; PTH, parathyroid hormone; R, right; ↓, decreased values; ↑, increased values.
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L. Yuan et al.: Identification of a new PHEX mutation in XLHR 29
I 1
2
II 1 N/M
2 N
3
4
5
6
4
5 N
6
7 N
III 1 N/M
2 N
1 N/M
2 M
3 N/N
IV
Figure 1 Pedigree of the family with X-linked hypophosphatemic rickets showing affected cases (fully shaded). N, normal; M, the PHEX p.F518Sfs*4 mutation.
Mutation screening We performed exome sequencing on genomic DNA sample of one patient (IV:1) of the Chinese Han family with HR. An average of 9.11 billion bases of sequence from the patient was generated as paired-end 90-bp reads. 8.89 billion bases (97.62%) passed the quality assessment, 8.35 billion bases (93.90%) were aligned to the human reference sequence and 4.35 billion bases were mapped to the target region with a mean coverage of 98.63-fold (Wang et al., 2011). 113,054 genetic variants, including 14,533 non-synonymous changes, were identified in the coding regions or
the canonical dinucleotide of the splice sites. A complete list of identified variants is represented in supplementary Table S1. A prioritization scheme was applied to identify the pathogenic mutation in the patient, similar to that in recent studies (Ng et al., 2010; Guo et al., 2014). Given that the frequency of HR is 0.50%. Using the above filtering criteria, we reduced the number of candidate genes by more than 88.62%. Sequence variants that were not annotated in any of the above public databases were further filtered by in-house database from BGI-Shenzhen with 2375 ethnically-matched controls, and only 274 variants were suspected to be novel. Subsequently, a deletion mutation in the PHEX gene, which is known to be the disease-causing gene of XLHR, was selected for further validation. After validation by Sanger sequencing and filtered from the PHEX locus database (http://www.phexdb. mcgill.ca/), a novel mutation, c.1553delT (p.F518Sfs*4), in the PHEX gene was observed in the patient. The same mutation was subsequently identified in all patients of this family (Figure 2). The mutation co-segregated with patients and was absent in unaffected individuals in this family and 100 ethnically-matched unrelated controls, suggesting that this variant is the pathogenic mutation. One hemizygous male patient (IV:2) and three heterozygous female patients (II:1, III:1 and IV:1) were found to carry the mutation. No obvious difference in severity of the disease between males and females was found, consistent with previous results (Francis et al., 1997; Holm et al., 2001; Cho et al., 2005). This frameshift mutation is located in exon 14, which encodes part of the large extracellular domain of the protein. The mutation leads to premature truncation at amino acid position 521 and disrupts the
Figure 2 Sequence analysis of the p.F518Sfs*4 mutation in the PHEX gene (DNA). (A) Unaffected member (III:3) of the family. (B) Heterozygous p.F518Sfs*4 mutation patient (III:1). (C) Hemizygous p.F518Sfs*4 mutation patient (IV:2).
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30 L. Yuan et al.: Identification of a new PHEX mutation in XLHR overall integrity of the PHEX protein predicted to result in loss of function (Gaucher et al., 2009; Kang et al., 2012).
Discussion Familial HR is a rare disorder with marked genetic heterogenecity and high clinical variability (Gaucher et al., 2009; Santos et al., 2013). XLHR, initially referred to as vitamin D-resistant rickets, is the most common form of HR, accounting for approximately 80% of the familial cases. XLHR is the prototypic disorder of FGF23-mediated hypophosphatemia (Baroncelli et al., 2012; Carpenter, 2012). The main clinical features of patients with XLHR are growth failure with disproportionate short stature, rickets, and lower extremity deformities. Patients also show some peculiar dental and periodontal abnormalities, and some features of muscle weakness (Baroncelli et al., 2012). The hallmark biochemical findings include hypophosphatemia, low or inappropriately normal circulating 1,25(OH)2D levels, elevated FGF23, decreased tubular threshold maximum for phosphate per glomerular filtration rate, elevated (in children) or normal (in adults) serum ALP activity, normal or elevated PTH, normal serum calcium, elevated urinary phosphate excretion, and normal or decreased urinary calcium excretion (Morey et al., 2011; Durmaz et al., 2013; Santos et al., 2013). Here we studied a Chinese Han family with hereditary rickets and variable other clinical features. Patients in this family were suspected clinically to have XLHR because of the pattern of disease transmission, such as lack of maleto-male transmission. However, autosomal dominant inheritance pattern cannot be excluded. All the patients have similar features, such as short stature, bowing of legs, hypophosphatemia, normal serum calcium, and elevated ALP and PTH levels. Hearing loss was found in two patients, consistent with previous studies, although an epiphenomenon or disease process of XLHR itself cannot be ruled out (Fishman et al., 2004; Kienitz et al., 2011). To our knowledge, the earliest onset age of bilateral mixed hearing loss was discovered in a 13-year-old girl (IV:1) who had no history of exposure to ototoxic drugs, noise or other chronic ear disease in our family (Fishman et al., 2004), and molecular mechanisms or pathways of incomplete penetrance in PHEX-related HR deafness await further investigations. Phenotypic variability increased the complexity of identifying the genetic cause of the disease. In this study, exome sequencing revealed a deletion of thymine (T) at nucleotide 1553 (p.F518Sfs*4) in the PHEX gene in the
tested patient (IV:1). Sanger sequencing showed that the variant co-segregated with patients, and was absent in unaffected individuals in the family, 100 normal controls, the PHEX locus database, dbSNP137, 1000 genome project, HapMap, and YanHuang project, suggesting this variant is the pathogenic mutation and confirming the diagnosis of XLHR in this family. XLHR is caused by inactivating mutations in the PHEX gene (Baroncelli et al., 2012). The PHEX gene, spanning ∼215 kb and consisting of 22 exons with huge intronic regions, is located at chromosome Xp22.1. The gene encodes a 749-amino acid protein, initially thought to directly inactivate FGF23 regulating the phosphate homeostasis (Morey et al., 2011; Durmaz et al., 2013; Santos et al., 2013). The PHEX protein consists of a short aminoterminal intracellular tail and transmembrane domain, and a large carboxyterminal extracellular region with the catalytic and zinc-binding sites (Morey et al., 2011). It belongs to the type II integral membrane zinc-dependent endopeptidase family and functions as a transmembrane endopeptidase, involved in bone and dentin mineralization and renal phosphate reabsorption (Morey et al., 2011; Durmaz et al., 2013; Fahiminiya et al., 2014). PHEX protein is expressed in late embryonic development as skeletal mineralization begins, with relatively high level in osteoblast, osteocytes and odontoblast, and localizes to the cell surface (Carpenter et al., 2011; Morey et al., 2011). At least 329 different mutations/polymorphisms in the PHEX gene have been described in the PHEX locus database (http://www.phexdb.mcgill.ca/, accessed March 2014). The majority of these mutations are found in XLHR patients. The PHEX mutations vary from point mutations to complex rearrangements, including frameshifts (25%), abnormal splicing mutations (23%), missense mutations (22%), nonsense mutations (18%), deletions (8%), as well as polymorphisms (4%). These mutations scatter throughout the PHEX gene without any obvious mutational hotspot, and most of them belong to the loss-offunction type (Holm et al., 2001; Kienitz et al., 2011; Kang et al., 2012). Pseudoexons, mosaic mutations and noncoding or intronic mutations have also been described (Dixon et al., 1998; Cho et al., 2005; Goji et al., 2006; Gaucher et al., 2009). No obvious genotype-phenotype correlation or genetic dosage effect has been found in most studies (Holm et al., 2001; Cho et al., 2005; Song et al., 2007). However, in a relatively small sample size, a trend toward more severe skeletal disease in familial patients with truncating mutations was reported (Holm et al., 2001). The PHEX gene is subject to random X chromosome inactivation (XCI) and some escape from inactivation has been reported (Holm et al., 2001; Song et al., 2007), and the
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L. Yuan et al.: Identification of a new PHEX mutation in XLHR 31
degree of XCI may contribute to the phenotypic variability. Skewed XCI that prevents the expression of the wild-type alleles may explain phenotypic similarity between heterozygous females and hemizygous males (Haack et al., 2012; Wang et al., 2013). The single-base deletion (c.1553delT) changed the codon at position 518, resulting in an amino acid substitution Phe518Ser located in the exon 14, and predicted to produce premature truncation at position 521 (p.F518Sfs*4). The predicted loss of the extracellular and zinc-binding domains of the mutation disrupts the overall integrity of the PHEX protein, which may lead to loss of protein function and hence leads to XLHR (Gaucher et al., 2009; Qiu et al., 2010; Kang et al., 2012). Mutation of the female patient (II:1) is thought to be a de novo mutation as none of her parents (I:1 and I:2) had any clinical evidence for HR, consistent with that de novo mutation is more frequent in females than males (Durmaz et al., 2013). De novo X-linked mutations occur predominantly on the malederived X chromosome, thus affecting only females. Male mutation bias, largely because of different processes of male and female gamete generation, is widespread among higher organisms and the ratio is about 6:1 in human (male:female). The frequent de novo mutations found in the female patients are likely resulting from mutagenesis of X chromosome in paternal germ cells (Zhang et al., 2012; Durmaz et al., 2013). To date, eight X-linked mutations (four spontaneous mutations, three ethylnitrosourea-generated mutations and a radiation-induced mutation) in the Phex gene have been identified in mice (Owen et al., 2012). These models manifest classic features of human XLHR, including growth retardation, skeletal deformities (rickets/osteomalacia), renal phosphate wasting, hypophosphatemia, elevated serum ALP level, and even variable expression of deafness (Lorenz-Depiereux et al., 2004; Owen et al., 2012). Mice with conditional inactivation of Phex in osteoblasts and osteocytes showed a phenotype similar to that in global Phex knockout mice, and comparable to that in Hyp (a large 3′ deletion of the Phex gene) mice, indicating aberrant Phex function in osteoblasts and/or osteocytes alone is sufficient to generate Hyp mouse phenotype (Yuan et al., 2008). Transgenic expression of the Phex or human PHEX (hPHEX) gene by osteoblast-specific promoters or the human β-actin promoter partially rescued the Hyp mouse phenotype (Erben et al., 2005), while osteoblast-specific expression of hPHEX gene could fully correct mineralization defects in 9-month-old Hyp mice (Boskey et al., 2009), and bone marrow transplantation improved abnormal bone mineral and vitamin D metabolism in Hyp mice (Miyamura et al., 2000), implying
osteoblast-involved target therapy would be a possible promising treatment for XLHR. In summary, our data support that the novel frameshift mutation, p.F518Sfs*4, is the genetic cause of XLHR in this family, and extend the spectrum of mutations in the PHEX gene. Whole exome sequencing provides a cost-effective and expedited approach. Applying it as a diagnostic tool may lead to unambiguous identification of diseasecausing mutations in phenotypically complex disorders (Fahiminiya et al., 2014). Our finding may provide new insights and approaches into the genetic cause and diagnosis of HR, and may also have implications for genetic counseling and clinical management. Further functional studies of the PHEX mutations and application of animal models with genetic deficiency may facilitate a better understanding of the pathogenesis and development of targeted experimental treatments of XLHR.
Materials and methods Participators and clinical evaluation A four-generation, seventeen-member Chinese Han family with familial HR was recruited from the Third Xiangya Hospital, Central South University, China (Figure 1). Clinical data and blood samples were obtained from nine members of the pedigree, including four affected individuals (II:1, III:1, IV:1 and IV:2) and five unaffected members (II:2, III:2, III:3, III:5 and III:7). All the available affected and unaffected individuals underwent detailed physical examinations, laboratory analyses, auditory and radiological examinations. Detailed medical and family histories were collected and recorded (Table 1). The diagnosis of familial HR was made based on positive family history of rickets, clinical manifestations, biochemical findings, and radiological evidence (Dixon et al., 1998; Gaucher et al., 2009; Kang et al., 2012). Blood samples were also collected from 100 unrelated ethnically-matched normal controls (male/female: 50/50; age 39.2 ± 8.3 years). Written informed consent was obtained from the participating individuals or their parents, and this study had received approval from the Ethics Committee of the Third Xiangya Hospital, Central South University, China.
Exome capture Genomic DNA was extracted from peripheral blood using standard phenol-chloroform extraction method (Guo et al., 2013). Three micrograms (μg) of genomic DNA was used to extract the exome library. Genomic DNA of one patient (IV:1) was sheared by sonication and then hybridized to the Nimblegen SeqCap EZ Library to enrich exonic DNA in each library, according to the manufacturer’s instructions. Sequencing of enriched library targeting the exome was performed on the Illumina HiSeq 2000 platform to generate 90-bp paired-end reads (Wang et al., 2011). A mean exome coverage of 78.87 × allowing
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32 L. Yuan et al.: Identification of a new PHEX mutation in XLHR each selected region of the genome to be checked was obtained. Such deep coverage provided sufficient depth to accurately call variants at 99.34% of targeted exome (Shi et al., 2011).
Read mapping and variant analysis The human reference genome was obtained from the available online UCSC database (http://genome.ucsc.edu/), version hg19 (build 37.1). Alignment of the sequences from the patient was performed using SOAPaligner (soap2.21) and SNPs were called using SOAPsnp set with the default parameters after the duplicated reads (obtained mainly in the PCR step) were deleted. Small insertions or deletions (indels) affecting coding sequence or splicing sites were detected (Li et al., 2008; Shi et al., 2011; Wang et al., 2011). The thresholds for calling SNPs and short indels included the number of unique mapped reads supporting a SNP ≥ 4 and the consensus quality score ≥ 20. The quality score is a Phred score, generated by the program SOAPsnp 1.05, quality = -10log (error rate). It is unlikely that causative variants present in the general population. All candidate variations identified in the subject were filtered against the Single Nucleotide Polymorphism database (dbSNP build 137, http://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi), 1000 genomes project (1000genomes release_20100804, http://www.1000genomes.org/), HapMap (201008_phase II + III, http://hapmap.ncbi.nlm.nih.gov/) and YanHuang (http://yh.genomics.org.cn/) project. Sorting intolerant from tolerant (SIFT) prediction (http://sift.jcvi.org/) was performed to estimate whether amino acid substitutions, amino acid insertions/deletions and frameshifting indels affect protein function (Wang et al., 2011; Hu and Ng, 2013).
Mutation validation Locus-specific PCR and detection primers were designed. Sanger sequencing was performed to validate the presence and identity of potential disease-causing variants with ABI3500 sequencer (Applied Biosystems Inc., Foster City, CA, USA). PCR amplification and Sanger sequencing were conducted as described previously (Yuan et al., 2013). Sequences of primers used for the PHEX gene causative variation were as follows: 5′-AGT TGC TCC TTC CTA TGC TGA-3′ and 5′-AGA CTC CGC TTC TCA CCA AT-3′.
Acknowledgments: We thank the participating members and investigators for their cooperation and efforts in collecting clinical and genetic information and DNA specimens. This work was supported by grants from National Natural Science Foundation of China (81271921; 81101339); Sheng Hua Scholars Program of Central South University, China (H.D.); Research Fund for the Doctoral Program of Higher Education of China (20110162110026); Science and Technology International Cooperation Key Project of Hunan Province, China (2011WK2011); Construction Fund for Key Subjects of the Third Xiangya Hospital, Central South University, China; Students Innovative Pilot Scheme of Central South University (YC12417), China; and
the Fundamental Research Funds for the Central Universities of Central South University, China (2013zzts101).
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Lamei Yuan, Song Wu, Hongbo Xu, Jingjing Xiao, Zhijian Yang, Hong Xia, An Liu, Pengzhi Hu, Anjie Lu, Yulan Chen, Fengping Xu and Hao Deng*
Identification of a novel PHEX mutation in a Chinese family with X-linked hypophosphatemic rickets using exome sequencing Abstract: Familial hypophosphatemic rickets (HR), the most common inherited form of rickets, is a group of inherited renal phosphate wasting disorders characterized by growth retardation, rickets with bone deformities, osteomalacia, poor dental development, and hypophosphatemia. The purpose of this study was to identify the genetic defect responsible for familial HR in a four-generation Chinese Han pedigree by exome sequencing and Sanger sequencing. Clinical features include skeletal deformities, teeth abnormalities, hearing impairments and variable serum phosphate level in patients of this family. A novel deletion mutation, c.1553delT (p.F518Sfs*4), was identified in the X-linked phosphate regulating endopeptidase homolog gene (PHEX). The mutation is predicted to result in prematurely truncated and loss-of-function PHEX protein. Our data suggest that exome sequencing is a powerful tool to discover mutation(s) in HR, a disorder with genetic and clinical heterogeneity. The findings may also provide new insights into the cause and diagnosis of HR, and have implications for genetic counseling and clinical management. Keywords: exome sequencing; frameshift; genetic counseling; hypophosphatemic rickets; PHEX gene.
*Corresponding author: Hao Deng, Center for Experimental Medicine, the Third Xiangya Hospital, Central South University, Changsha 410013, China; and Department of Neurology, the Third Xiangya Hospital, Central South University, Changsha 410013, China, e-mail: [email protected] Lamei Yuan, Hongbo Xu, Zhijian Yang and Hong Xia: Center for Experimental Medicine, the Third Xiangya Hospital, Central South University, Changsha 410013, China Song Wu and Anjie Lu: Department of Orthopedics, the Third Xiangya Hospital, Central South University, Changsha 410013, China Jingjing Xiao, Yulan Chen and Fengping Xu: BGI-Shenzhen, Shenzhen 518083, China An Liu: Department of Otolaryngology-Head Neck Surgery, the Third Xiangya Hospital, Central South University, Changsha 410013, China Pengzhi Hu: Department of Radiology, the Third Xiangya Hospital, Central South University, Changsha 410013, China
DOI 10.1515/hsz-2014-0187 Received April 17, 2014; accepted July 20, 2014; previously published online July 23, 2014
Introduction Familial hypophosphatemic rickets (HR) is the most common inherited form of rickets. It was first described by Albright et al. in 1937 (Goji et al., 2006; Baroncelli et al., 2012). Familial HR consists of a group of renal phosphate wasting disorders with hypophosphatemia and inappropriately low or normal serum 1,25-dihydroxyvitamin D (1,25(OH)2D) levels (Morey et al., 2011). Familial HR is characterized by marked clinical variability of growth retardation, childhood rickets, osteomalacia, and poor dental development (Dixon et al., 1998; Goji et al., 2006; Morey et al., 2011; Santos et al., 2013). In addition, extraskeletal bone formation, osteoarthritis, limitation of joint mobility, and occasionally spinal cord compression may occur (Dixon et al., 1998). It was reported that average incidence of HR is 3.9 per 100,000 in children between age 0 and 0.9 years, and a prevalence of 4.8 per 100,000 in those younger than 15 years (Beck-Nielsen et al., 2009). Six types of hereditary HR have been identified, including the common X-linked dominant HR (XLHR, MIM 307800), the occasional autosomal dominant HR (ADHR, MIM 193100), autosomal recessive HR (ARHR1, MIM 241520, and ARHR2, MIM 613312), the rare X-linked recessive HR (MIM 300554), and two peculiar rare forms of HR associated with hypercalciuria (HHRH, MIM 241530) and hyperparathyroidism (MIM 612089) (Morey et al., 2011; Baroncelli et al., 2012; Santos et al., 2013). HR may be caused by mutations of genes involved in the complex regulation of phosphate metabolism (Baroncelli et al., 2012). To date, at least seven disease loci and six disease-causing genes, including the X-linked phosphate regulating endopeptidase homolog gene (PHEX, MIM 300550), the fibroblast growth factor 23 gene (FGF23, MIM 605380), the dentin matrix acidic phosphoprotein 1 gene (DMP1, MIM 600980), the ectonucleotide pyrophosphatase/phosphodiesterase 1 gene (ENPP1,
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28 L. Yuan et al.: Identification of a new PHEX mutation in XLHR MIM 173335), the chloride channel voltage-sensitive 5 gene (CLCN5, MIM 300008) and the solute carrier family 34 (sodium/phosphate cotransporter) member 3 gene (SLC34A3, MIM 609826), have been reported (Baroncelli et al., 2012). Among them, XLHR is the most common form, and it is caused by inactivating mutations in the PHEX gene, which is associated with increased circulating FGF23 levels (Baroncelli et al., 2012; Santos et al., 2013). XLHR patients exhibit marked phenotypic variability, and some may have normal growth (Makras et al., 2008; Baroncelli et al., 2012). The main purpose of this study was to identify the gene responsible for familial HR in a four-generation Chinese pedigree. A novel frameshift mutation (c.1553delT, p.F518Sfs*4) in the PHEX gene, disrupting the overall integrity of the PHEX protein, was found to co-segregate with HR patients in this family and be absent in normal controls, indicating that it is a pathogenic mutation.
Results Clinical findings Detailed clinical, laboratory, auditory and radiological characteristics are summarized in Table 1. All patients
in the family had short stature and apparent lower extremity deformities. Blood biochemical tests revealed various degrees of low serum phosphate, normal serum calcium, elevated parathyoid hormone (PTH) and alkaline phosphatase (ALP) levels. Three patients complained of gait disturbance, bone pain and fatigue. A patient (II:1, Figure 1) lost all the teeth in her thirties, and her daughter (III:1) also complained about losing teeth in early adulthood. The 13-year-old proband (IV:1) had nearly normal tooth development, but suffered from hearing impairment. External auditory meatus and tympanic membrane of all patients were normal. Two patients (II:1 and IV:1) showed hearing impairments. One (II:1) was 62 years old, hence presbyacusis cannot be ruled out (Fishman et al., 2004). The other was a young female (IV:1) with bilateral mixed hearing loss. The severity of some clinical features, including bone pain, dental anomalies and hearing impairment varied among patients of this family. The proband was diagnosed with HR based on diagnostic criteria. According to the medical records, there were an additional three patients (II:1, III:1 and IV:2) who were also diagnosed with HR manifested disabilities in walking and severely growth retardation, and positive family history confirmed the diagnosis of familial HR in this family (Morey et al., 2011; Kang et al., 2012).
Table 1 Clinical, laboratory, auditory, radiological and genetic data of four patients with the PHEX c.1553delT mutation. Subject
II:1
Gender Age (years) Genotype Weight (kg) Height (cm) Growth retardation Bowing of legs Disabilities in walking Bone pain Skeletal deformities Dental anomalies Hearing impairment
Serum phosphate Serum calcium Serum ALP Serum PTH Urine phosphate Urine calcium Radiological changes
III:1
Female 62 Heterozygote 46 122 Yes Yes Yes Yes Yes Premature tooth loss, edentulous jaw Sensorineural hearing loss (R), mixed hearing loss (L) ↓ N ↑ ↑ ↑ ↓ Yes
IV:1
Female 33 Heterozygote 52 117 Yes Yes Yes Yes Yes Premature tooth loss, periodontitis No ↓ N ↑ ↑ ↑ ↓ Yes
IV:2
Female 13 Heterozygote 27 116 Yes Yes Yes Yes Yes Enamel hypoplasia Bilateral mixed hearing loss ↓ N ↑ ↑ ↑ ↓ Yes
Male 6 Hemizygote 17 68 Yes Yes Yes No Yes Hypodontia, enamel hypoplasia, prematureloss deciduous tooth, dental caries N/A ↓ N ↑ ↑ ↑ ↓ Yes
ALP, alkaline phosphatase; L, left; N, normal values; N/A, not available; PHEX, the X-linked phosphate regulating endopeptidase homolog gene; PTH, parathyroid hormone; R, right; ↓, decreased values; ↑, increased values.
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L. Yuan et al.: Identification of a new PHEX mutation in XLHR 29
I 1
2
II 1 N/M
2 N
3
4
5
6
4
5 N
6
7 N
III 1 N/M
2 N
1 N/M
2 M
3 N/N
IV
Figure 1 Pedigree of the family with X-linked hypophosphatemic rickets showing affected cases (fully shaded). N, normal; M, the PHEX p.F518Sfs*4 mutation.
Mutation screening We performed exome sequencing on genomic DNA sample of one patient (IV:1) of the Chinese Han family with HR. An average of 9.11 billion bases of sequence from the patient was generated as paired-end 90-bp reads. 8.89 billion bases (97.62%) passed the quality assessment, 8.35 billion bases (93.90%) were aligned to the human reference sequence and 4.35 billion bases were mapped to the target region with a mean coverage of 98.63-fold (Wang et al., 2011). 113,054 genetic variants, including 14,533 non-synonymous changes, were identified in the coding regions or
the canonical dinucleotide of the splice sites. A complete list of identified variants is represented in supplementary Table S1. A prioritization scheme was applied to identify the pathogenic mutation in the patient, similar to that in recent studies (Ng et al., 2010; Guo et al., 2014). Given that the frequency of HR is 0.50%. Using the above filtering criteria, we reduced the number of candidate genes by more than 88.62%. Sequence variants that were not annotated in any of the above public databases were further filtered by in-house database from BGI-Shenzhen with 2375 ethnically-matched controls, and only 274 variants were suspected to be novel. Subsequently, a deletion mutation in the PHEX gene, which is known to be the disease-causing gene of XLHR, was selected for further validation. After validation by Sanger sequencing and filtered from the PHEX locus database (http://www.phexdb. mcgill.ca/), a novel mutation, c.1553delT (p.F518Sfs*4), in the PHEX gene was observed in the patient. The same mutation was subsequently identified in all patients of this family (Figure 2). The mutation co-segregated with patients and was absent in unaffected individuals in this family and 100 ethnically-matched unrelated controls, suggesting that this variant is the pathogenic mutation. One hemizygous male patient (IV:2) and three heterozygous female patients (II:1, III:1 and IV:1) were found to carry the mutation. No obvious difference in severity of the disease between males and females was found, consistent with previous results (Francis et al., 1997; Holm et al., 2001; Cho et al., 2005). This frameshift mutation is located in exon 14, which encodes part of the large extracellular domain of the protein. The mutation leads to premature truncation at amino acid position 521 and disrupts the
Figure 2 Sequence analysis of the p.F518Sfs*4 mutation in the PHEX gene (DNA). (A) Unaffected member (III:3) of the family. (B) Heterozygous p.F518Sfs*4 mutation patient (III:1). (C) Hemizygous p.F518Sfs*4 mutation patient (IV:2).
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30 L. Yuan et al.: Identification of a new PHEX mutation in XLHR overall integrity of the PHEX protein predicted to result in loss of function (Gaucher et al., 2009; Kang et al., 2012).
Discussion Familial HR is a rare disorder with marked genetic heterogenecity and high clinical variability (Gaucher et al., 2009; Santos et al., 2013). XLHR, initially referred to as vitamin D-resistant rickets, is the most common form of HR, accounting for approximately 80% of the familial cases. XLHR is the prototypic disorder of FGF23-mediated hypophosphatemia (Baroncelli et al., 2012; Carpenter, 2012). The main clinical features of patients with XLHR are growth failure with disproportionate short stature, rickets, and lower extremity deformities. Patients also show some peculiar dental and periodontal abnormalities, and some features of muscle weakness (Baroncelli et al., 2012). The hallmark biochemical findings include hypophosphatemia, low or inappropriately normal circulating 1,25(OH)2D levels, elevated FGF23, decreased tubular threshold maximum for phosphate per glomerular filtration rate, elevated (in children) or normal (in adults) serum ALP activity, normal or elevated PTH, normal serum calcium, elevated urinary phosphate excretion, and normal or decreased urinary calcium excretion (Morey et al., 2011; Durmaz et al., 2013; Santos et al., 2013). Here we studied a Chinese Han family with hereditary rickets and variable other clinical features. Patients in this family were suspected clinically to have XLHR because of the pattern of disease transmission, such as lack of maleto-male transmission. However, autosomal dominant inheritance pattern cannot be excluded. All the patients have similar features, such as short stature, bowing of legs, hypophosphatemia, normal serum calcium, and elevated ALP and PTH levels. Hearing loss was found in two patients, consistent with previous studies, although an epiphenomenon or disease process of XLHR itself cannot be ruled out (Fishman et al., 2004; Kienitz et al., 2011). To our knowledge, the earliest onset age of bilateral mixed hearing loss was discovered in a 13-year-old girl (IV:1) who had no history of exposure to ototoxic drugs, noise or other chronic ear disease in our family (Fishman et al., 2004), and molecular mechanisms or pathways of incomplete penetrance in PHEX-related HR deafness await further investigations. Phenotypic variability increased the complexity of identifying the genetic cause of the disease. In this study, exome sequencing revealed a deletion of thymine (T) at nucleotide 1553 (p.F518Sfs*4) in the PHEX gene in the
tested patient (IV:1). Sanger sequencing showed that the variant co-segregated with patients, and was absent in unaffected individuals in the family, 100 normal controls, the PHEX locus database, dbSNP137, 1000 genome project, HapMap, and YanHuang project, suggesting this variant is the pathogenic mutation and confirming the diagnosis of XLHR in this family. XLHR is caused by inactivating mutations in the PHEX gene (Baroncelli et al., 2012). The PHEX gene, spanning ∼215 kb and consisting of 22 exons with huge intronic regions, is located at chromosome Xp22.1. The gene encodes a 749-amino acid protein, initially thought to directly inactivate FGF23 regulating the phosphate homeostasis (Morey et al., 2011; Durmaz et al., 2013; Santos et al., 2013). The PHEX protein consists of a short aminoterminal intracellular tail and transmembrane domain, and a large carboxyterminal extracellular region with the catalytic and zinc-binding sites (Morey et al., 2011). It belongs to the type II integral membrane zinc-dependent endopeptidase family and functions as a transmembrane endopeptidase, involved in bone and dentin mineralization and renal phosphate reabsorption (Morey et al., 2011; Durmaz et al., 2013; Fahiminiya et al., 2014). PHEX protein is expressed in late embryonic development as skeletal mineralization begins, with relatively high level in osteoblast, osteocytes and odontoblast, and localizes to the cell surface (Carpenter et al., 2011; Morey et al., 2011). At least 329 different mutations/polymorphisms in the PHEX gene have been described in the PHEX locus database (http://www.phexdb.mcgill.ca/, accessed March 2014). The majority of these mutations are found in XLHR patients. The PHEX mutations vary from point mutations to complex rearrangements, including frameshifts (25%), abnormal splicing mutations (23%), missense mutations (22%), nonsense mutations (18%), deletions (8%), as well as polymorphisms (4%). These mutations scatter throughout the PHEX gene without any obvious mutational hotspot, and most of them belong to the loss-offunction type (Holm et al., 2001; Kienitz et al., 2011; Kang et al., 2012). Pseudoexons, mosaic mutations and noncoding or intronic mutations have also been described (Dixon et al., 1998; Cho et al., 2005; Goji et al., 2006; Gaucher et al., 2009). No obvious genotype-phenotype correlation or genetic dosage effect has been found in most studies (Holm et al., 2001; Cho et al., 2005; Song et al., 2007). However, in a relatively small sample size, a trend toward more severe skeletal disease in familial patients with truncating mutations was reported (Holm et al., 2001). The PHEX gene is subject to random X chromosome inactivation (XCI) and some escape from inactivation has been reported (Holm et al., 2001; Song et al., 2007), and the
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L. Yuan et al.: Identification of a new PHEX mutation in XLHR 31
degree of XCI may contribute to the phenotypic variability. Skewed XCI that prevents the expression of the wild-type alleles may explain phenotypic similarity between heterozygous females and hemizygous males (Haack et al., 2012; Wang et al., 2013). The single-base deletion (c.1553delT) changed the codon at position 518, resulting in an amino acid substitution Phe518Ser located in the exon 14, and predicted to produce premature truncation at position 521 (p.F518Sfs*4). The predicted loss of the extracellular and zinc-binding domains of the mutation disrupts the overall integrity of the PHEX protein, which may lead to loss of protein function and hence leads to XLHR (Gaucher et al., 2009; Qiu et al., 2010; Kang et al., 2012). Mutation of the female patient (II:1) is thought to be a de novo mutation as none of her parents (I:1 and I:2) had any clinical evidence for HR, consistent with that de novo mutation is more frequent in females than males (Durmaz et al., 2013). De novo X-linked mutations occur predominantly on the malederived X chromosome, thus affecting only females. Male mutation bias, largely because of different processes of male and female gamete generation, is widespread among higher organisms and the ratio is about 6:1 in human (male:female). The frequent de novo mutations found in the female patients are likely resulting from mutagenesis of X chromosome in paternal germ cells (Zhang et al., 2012; Durmaz et al., 2013). To date, eight X-linked mutations (four spontaneous mutations, three ethylnitrosourea-generated mutations and a radiation-induced mutation) in the Phex gene have been identified in mice (Owen et al., 2012). These models manifest classic features of human XLHR, including growth retardation, skeletal deformities (rickets/osteomalacia), renal phosphate wasting, hypophosphatemia, elevated serum ALP level, and even variable expression of deafness (Lorenz-Depiereux et al., 2004; Owen et al., 2012). Mice with conditional inactivation of Phex in osteoblasts and osteocytes showed a phenotype similar to that in global Phex knockout mice, and comparable to that in Hyp (a large 3′ deletion of the Phex gene) mice, indicating aberrant Phex function in osteoblasts and/or osteocytes alone is sufficient to generate Hyp mouse phenotype (Yuan et al., 2008). Transgenic expression of the Phex or human PHEX (hPHEX) gene by osteoblast-specific promoters or the human β-actin promoter partially rescued the Hyp mouse phenotype (Erben et al., 2005), while osteoblast-specific expression of hPHEX gene could fully correct mineralization defects in 9-month-old Hyp mice (Boskey et al., 2009), and bone marrow transplantation improved abnormal bone mineral and vitamin D metabolism in Hyp mice (Miyamura et al., 2000), implying
osteoblast-involved target therapy would be a possible promising treatment for XLHR. In summary, our data support that the novel frameshift mutation, p.F518Sfs*4, is the genetic cause of XLHR in this family, and extend the spectrum of mutations in the PHEX gene. Whole exome sequencing provides a cost-effective and expedited approach. Applying it as a diagnostic tool may lead to unambiguous identification of diseasecausing mutations in phenotypically complex disorders (Fahiminiya et al., 2014). Our finding may provide new insights and approaches into the genetic cause and diagnosis of HR, and may also have implications for genetic counseling and clinical management. Further functional studies of the PHEX mutations and application of animal models with genetic deficiency may facilitate a better understanding of the pathogenesis and development of targeted experimental treatments of XLHR.
Materials and methods Participators and clinical evaluation A four-generation, seventeen-member Chinese Han family with familial HR was recruited from the Third Xiangya Hospital, Central South University, China (Figure 1). Clinical data and blood samples were obtained from nine members of the pedigree, including four affected individuals (II:1, III:1, IV:1 and IV:2) and five unaffected members (II:2, III:2, III:3, III:5 and III:7). All the available affected and unaffected individuals underwent detailed physical examinations, laboratory analyses, auditory and radiological examinations. Detailed medical and family histories were collected and recorded (Table 1). The diagnosis of familial HR was made based on positive family history of rickets, clinical manifestations, biochemical findings, and radiological evidence (Dixon et al., 1998; Gaucher et al., 2009; Kang et al., 2012). Blood samples were also collected from 100 unrelated ethnically-matched normal controls (male/female: 50/50; age 39.2 ± 8.3 years). Written informed consent was obtained from the participating individuals or their parents, and this study had received approval from the Ethics Committee of the Third Xiangya Hospital, Central South University, China.
Exome capture Genomic DNA was extracted from peripheral blood using standard phenol-chloroform extraction method (Guo et al., 2013). Three micrograms (μg) of genomic DNA was used to extract the exome library. Genomic DNA of one patient (IV:1) was sheared by sonication and then hybridized to the Nimblegen SeqCap EZ Library to enrich exonic DNA in each library, according to the manufacturer’s instructions. Sequencing of enriched library targeting the exome was performed on the Illumina HiSeq 2000 platform to generate 90-bp paired-end reads (Wang et al., 2011). A mean exome coverage of 78.87 × allowing
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32 L. Yuan et al.: Identification of a new PHEX mutation in XLHR each selected region of the genome to be checked was obtained. Such deep coverage provided sufficient depth to accurately call variants at 99.34% of targeted exome (Shi et al., 2011).
Read mapping and variant analysis The human reference genome was obtained from the available online UCSC database (http://genome.ucsc.edu/), version hg19 (build 37.1). Alignment of the sequences from the patient was performed using SOAPaligner (soap2.21) and SNPs were called using SOAPsnp set with the default parameters after the duplicated reads (obtained mainly in the PCR step) were deleted. Small insertions or deletions (indels) affecting coding sequence or splicing sites were detected (Li et al., 2008; Shi et al., 2011; Wang et al., 2011). The thresholds for calling SNPs and short indels included the number of unique mapped reads supporting a SNP ≥ 4 and the consensus quality score ≥ 20. The quality score is a Phred score, generated by the program SOAPsnp 1.05, quality = -10log (error rate). It is unlikely that causative variants present in the general population. All candidate variations identified in the subject were filtered against the Single Nucleotide Polymorphism database (dbSNP build 137, http://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi), 1000 genomes project (1000genomes release_20100804, http://www.1000genomes.org/), HapMap (201008_phase II + III, http://hapmap.ncbi.nlm.nih.gov/) and YanHuang (http://yh.genomics.org.cn/) project. Sorting intolerant from tolerant (SIFT) prediction (http://sift.jcvi.org/) was performed to estimate whether amino acid substitutions, amino acid insertions/deletions and frameshifting indels affect protein function (Wang et al., 2011; Hu and Ng, 2013).
Mutation validation Locus-specific PCR and detection primers were designed. Sanger sequencing was performed to validate the presence and identity of potential disease-causing variants with ABI3500 sequencer (Applied Biosystems Inc., Foster City, CA, USA). PCR amplification and Sanger sequencing were conducted as described previously (Yuan et al., 2013). Sequences of primers used for the PHEX gene causative variation were as follows: 5′-AGT TGC TCC TTC CTA TGC TGA-3′ and 5′-AGA CTC CGC TTC TCA CCA AT-3′.
Acknowledgments: We thank the participating members and investigators for their cooperation and efforts in collecting clinical and genetic information and DNA specimens. This work was supported by grants from National Natural Science Foundation of China (81271921; 81101339); Sheng Hua Scholars Program of Central South University, China (H.D.); Research Fund for the Doctoral Program of Higher Education of China (20110162110026); Science and Technology International Cooperation Key Project of Hunan Province, China (2011WK2011); Construction Fund for Key Subjects of the Third Xiangya Hospital, Central South University, China; Students Innovative Pilot Scheme of Central South University (YC12417), China; and
the Fundamental Research Funds for the Central Universities of Central South University, China (2013zzts101).
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