title: Whole Exome Sequencing Reveals Novel PHEX Splice Site Mutations in Patients with Hypophosphatemic Rickets.
abstract: OBJECTIVE
  Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features.
  
  
  METHODS
  We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family.
  
  
  RESULTS
  WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient's EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human exons. Direct Sanger sequencing of all PHEX exons in a sporadic case with HR from the Indian subcontinent revealed an additional novel PHEX mutation (c.1211_1215delACAAAinsTTTACAT, p.Asp404Valfs*5, de novo) located in exon 11.
  
  
  CONCLUSIONS
  Mutation analyses revealed two novel mutations and helped to confirm the clinical diagnoses of XLH in two families from India. WES helped to analyze all genes implicated in the underlying disease complex. Mutations at splice positions other than the canonical key sites need further functional investigation to support the assertion of pathogenicity.
authors.affiliation: College of Literature, Science, and the Arts, University of Michigan, Ann Arbor, MI, United States of America.
authors.forename: Sara L
authors.initials: SL
authors.lastname: Ma
authors.affiliation: Division of Nephrology, Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, United States of America.
authors.forename: Virginia
authors.initials: V
authors.lastname: Vega-Warner
authors.affiliation: Division of Nephrology, Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, United States of America.
authors.forename: Christopher
authors.initials: C
authors.lastname: Gillies
authors.affiliation: Division of Nephrology, Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, United States of America.
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Sampson
authors.affiliation: Kidney and Urology Institute, Medanta, The Medicity Hospital, Gurgaon, India.
authors.forename: Vijay
authors.initials: V
authors.lastname: Kher
authors.affiliation: Kidney and Urology Institute, Medanta, The Medicity Hospital, Gurgaon, India.
authors.forename: Sidharth K
authors.initials: SK
authors.lastname: Sethi
authors.affiliation: Division of Nephrology, Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, United States of America.
authors.forename: Edgar A
authors.initials: EA
authors.lastname: Otto
mesh_terms: D000328:Adult; D017398:Alternative Splicing; D001482:Base Composition; D002461:Cell Line, Transformed; D002648:Child; D041321:Chromosomes, Human, X / Q000235:genetics; D003198:Computer Simulation; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D059472:Exome / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D053098:Familial Hypophosphatemic Rickets / Q000175:diagnosis / Q000235:genetics*; D005260:Female; D000089703:Fibroblast Growth Factor-23; D040181:Genetic Diseases, X-Linked / Q000175:diagnosis / Q000235:genetics*; D006801:Humans; D007194:India; D007438:Introns / Q000235:genetics*; D008297:Male; D053402:PHEX Phosphate Regulating Neutral Endopeptidase / Q000737:chemistry / Q000235:genetics*; D010375:Pedigree; D017354:Point Mutation; D022821:RNA Splice Sites / Q000235:genetics*; D063730:Rickets, Hypophosphatemic / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: C000717427:FGF23 protein, human; D022821:RNA Splice Sites; D000089703:Fibroblast Growth Factor-23; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; C506231:PHEX protein, human