J Pediatr Endocr Met 2015; 28(1-2): 211–216
Patient report Minjing Zou, Derya Buluşa, Roua A. Al-Rijjal, Nesibe Andıran, Huda BinEssa, Walaa E. Kattan, Brian Meyer and Yufei Shi*
Hypophosphatemic rickets caused by a novel splice donor site mutation and activation of two cryptic splice donor sites in the PHEX gene Abstract: X-linked hypophosphatemic rickets (XLH) is the most common inherited form of rickets. XLH is caused by inactivating mutations in the PHEX gene and is transmitted as an X-linked dominant disorder. We investigated PHEX mutation in a sporadic Turkish girl with hypophosphatemic rickets. The patient was 2 years of age with a complaint of inability to walk. She had bowing of legs and growth retardation. Laboratory data showed normal calcium, low phosphate with markedly elevated ALP, and low phosphate renal tubular reabsorption. She was treated with Calcitriol 0.5 mg/kg/day and oral phosphate supplement with good response. The entire coding region of PHEX gene was sequenced from patient’s peripheral leukocyte DNA and a novel 13 bp deletion at the donor splice site of exon5 was found (c.663+12del). Instead of using the donor splice site of intron 4 to splice out exon 5 and intron 5, the spliceosome utilized two nearby cryptic donor splice sites (5′ splice site) to splice out intron 4, resulting in two smaller transcripts. Both of them could not translate into functional proteins due to frameshift. Her parents did not carry the mutation, indicating that this is a de novo PHEX mutation likely resulting from mutagenesis of X chromosome in paternal germ cells. We conclude that c.663+12del is a novel mutation that can activate nearby cryptic 5′ splice sites. The selection of cryptic 5′ splice sites adds the complexity of cell’s splicing mechanisms. The current study extends the database of PHEX mutation and cryptic 5′ splice sites. Contribute equally to the work. *Corresponding author: Yufei Shi, MD, Department of Genetics (MBC-03), King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, E-mail: [email protected] Minjing Zou, Roua A. Al-Rijjal, Huda BinEssa and Brian Meyer: Department of Genetics (MBC-03), King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia Walaa E. Kattan: College of Science and General Studies, Alfaisal University, Riyadh Derya Buluş and Nesibe Andıran: Department of Pediatric Endocrinology, Keçiören Research and Educational Hospital, Ankara, Turkey a
Keywords: hypophosphatemic rickets; PHEX mutation; vitamin D; X-linked. DOI 10.1515/jpem-2014-0103 Received February 27, 2014; accepted June 19, 2014; previously published online August 5, 2014
Introduction Hypophosphatemic rickets (HR) or vitamin D-resistant rickets is a group of renal phosphate wasting disorders including X-linked hypophosphatemic rickets (XLH), also called X-linked dominant hypophosphatemic rickets, autosomal dominant hypophosphatemic rickets, or autosomal recessive hypophosphatemic rickets. They result from defects in renal tubular reabsorption of phosphate and share similar clinical and biochemical features, such as bone deformity, bone pain, short stature, poor dental development, and hypophosphatemia with inappropriately low or normal 1,25-dihydroxyvitamin D3 (1,25(OH)2D) serum levels (1). X-linked hypophosphatemic rickets is the most common form with an incidence of approximately 1 in 20,000 live births (2). XLH is caused by inactivating mutations in the PHEX gene (phosphate regulating gene with homologies to endopeptidases on the X chromosome) (3). The PHEX gene is located on X chromosome Xp22.1 and encodes for a 749 amino acid glycoprotein with 22 exons (4). It has characteristic features of type II membrane glycoproteins: a short N-terminal cytoplasmic domain, a single transmembrane region, and a large extracellular domain (5). It functions as a transmembrane endopeptidase and is expressed primarily in cells of bone lineage, such as osteoblasts, osteocytes, and odontoblasts (6). To date, more than 329 different PHEX mutations have been described in patients with hypophosphatemic rickets, which include nonsense mutations, missense mutations,
Brought to you by | University of California - San Francisco Authenticated Download Date | 2/14/15 2:22 PM
212 Zou et al.: PHEX gene mutation in hypophosphatemic rickets splicing site mutations, insertions, and deletions in different positions (http://www.phexdb.mcgill.ca/) (7, 8). We previously reported four novel PHEX mutations in Turkish patients with hypophosphatemic rickets (9). In the present study, we reported another novel PHEX mutation in a Turkish girl with sporadic hypophosphatemic rickets.
(reverse primer located in exon 6). The resulting PCR products were reamplified by PCR using internal forward primer (5′-ATTCATCCTGCATGAATGAGA-3′ in exon 4) and the reverse primer. The PCR conditions were 95°C for 5 min followed by 35 cycles of amplification (95°C for 40 s, 50°C for 40 s, and 72°C for 40 s). The resulting PCR products were analyzed by gel electrophoresis and cloned into TA vector (Invitrogen, CA, USA). Individual clones were subsequently sequenced.
Subjects and methods
Donor splice site prediction
The patient was a 2-year-old girl with a complaint of inability to walk. She had classical features of rickets, such as frontal bossing, widened epiphysis at wrist, rickety rosary, and bowed legs. She also had growth retardation: her height was 77 cm (height standard deviation score: –2.82,
Patient report Minjing Zou, Derya Buluşa, Roua A. Al-Rijjal, Nesibe Andıran, Huda BinEssa, Walaa E. Kattan, Brian Meyer and Yufei Shi*
Hypophosphatemic rickets caused by a novel splice donor site mutation and activation of two cryptic splice donor sites in the PHEX gene Abstract: X-linked hypophosphatemic rickets (XLH) is the most common inherited form of rickets. XLH is caused by inactivating mutations in the PHEX gene and is transmitted as an X-linked dominant disorder. We investigated PHEX mutation in a sporadic Turkish girl with hypophosphatemic rickets. The patient was 2 years of age with a complaint of inability to walk. She had bowing of legs and growth retardation. Laboratory data showed normal calcium, low phosphate with markedly elevated ALP, and low phosphate renal tubular reabsorption. She was treated with Calcitriol 0.5 mg/kg/day and oral phosphate supplement with good response. The entire coding region of PHEX gene was sequenced from patient’s peripheral leukocyte DNA and a novel 13 bp deletion at the donor splice site of exon5 was found (c.663+12del). Instead of using the donor splice site of intron 4 to splice out exon 5 and intron 5, the spliceosome utilized two nearby cryptic donor splice sites (5′ splice site) to splice out intron 4, resulting in two smaller transcripts. Both of them could not translate into functional proteins due to frameshift. Her parents did not carry the mutation, indicating that this is a de novo PHEX mutation likely resulting from mutagenesis of X chromosome in paternal germ cells. We conclude that c.663+12del is a novel mutation that can activate nearby cryptic 5′ splice sites. The selection of cryptic 5′ splice sites adds the complexity of cell’s splicing mechanisms. The current study extends the database of PHEX mutation and cryptic 5′ splice sites. Contribute equally to the work. *Corresponding author: Yufei Shi, MD, Department of Genetics (MBC-03), King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, E-mail: [email protected] Minjing Zou, Roua A. Al-Rijjal, Huda BinEssa and Brian Meyer: Department of Genetics (MBC-03), King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia Walaa E. Kattan: College of Science and General Studies, Alfaisal University, Riyadh Derya Buluş and Nesibe Andıran: Department of Pediatric Endocrinology, Keçiören Research and Educational Hospital, Ankara, Turkey a
Keywords: hypophosphatemic rickets; PHEX mutation; vitamin D; X-linked. DOI 10.1515/jpem-2014-0103 Received February 27, 2014; accepted June 19, 2014; previously published online August 5, 2014
Introduction Hypophosphatemic rickets (HR) or vitamin D-resistant rickets is a group of renal phosphate wasting disorders including X-linked hypophosphatemic rickets (XLH), also called X-linked dominant hypophosphatemic rickets, autosomal dominant hypophosphatemic rickets, or autosomal recessive hypophosphatemic rickets. They result from defects in renal tubular reabsorption of phosphate and share similar clinical and biochemical features, such as bone deformity, bone pain, short stature, poor dental development, and hypophosphatemia with inappropriately low or normal 1,25-dihydroxyvitamin D3 (1,25(OH)2D) serum levels (1). X-linked hypophosphatemic rickets is the most common form with an incidence of approximately 1 in 20,000 live births (2). XLH is caused by inactivating mutations in the PHEX gene (phosphate regulating gene with homologies to endopeptidases on the X chromosome) (3). The PHEX gene is located on X chromosome Xp22.1 and encodes for a 749 amino acid glycoprotein with 22 exons (4). It has characteristic features of type II membrane glycoproteins: a short N-terminal cytoplasmic domain, a single transmembrane region, and a large extracellular domain (5). It functions as a transmembrane endopeptidase and is expressed primarily in cells of bone lineage, such as osteoblasts, osteocytes, and odontoblasts (6). To date, more than 329 different PHEX mutations have been described in patients with hypophosphatemic rickets, which include nonsense mutations, missense mutations,
Brought to you by | University of California - San Francisco Authenticated Download Date | 2/14/15 2:22 PM
212 Zou et al.: PHEX gene mutation in hypophosphatemic rickets splicing site mutations, insertions, and deletions in different positions (http://www.phexdb.mcgill.ca/) (7, 8). We previously reported four novel PHEX mutations in Turkish patients with hypophosphatemic rickets (9). In the present study, we reported another novel PHEX mutation in a Turkish girl with sporadic hypophosphatemic rickets.
(reverse primer located in exon 6). The resulting PCR products were reamplified by PCR using internal forward primer (5′-ATTCATCCTGCATGAATGAGA-3′ in exon 4) and the reverse primer. The PCR conditions were 95°C for 5 min followed by 35 cycles of amplification (95°C for 40 s, 50°C for 40 s, and 72°C for 40 s). The resulting PCR products were analyzed by gel electrophoresis and cloned into TA vector (Invitrogen, CA, USA). Individual clones were subsequently sequenced.
Subjects and methods
Donor splice site prediction
The patient was a 2-year-old girl with a complaint of inability to walk. She had classical features of rickets, such as frontal bossing, widened epiphysis at wrist, rickety rosary, and bowed legs. She also had growth retardation: her height was 77 cm (height standard deviation score: –2.82,
