Whole Exome Sequencing Reveals Novel PHEX Splice Site Mutations in Patients with Hypophosphatemic Rickets

Abstract

ObjectiveHypophosphatemic 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.MethodsWe 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.ResultsWES 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.ConclusionsMutation 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.

Clinical practice recommendations for the diagnosis and management of X-linked hypophosphataemia

“…3; TABle 2). Extended molecular genetic analysis can be helpful to establish the diagnosis in unclear cases of hypophosphataemic rickets [69][70][71] . Nutritional rickets and XLH might sometimes coexist, and diagnosis of XLH should be considered in someone otherwise thought to be vitamin D or calcium deficient if serum levels of phosphate do not improve after supplementation.…”

Genetics of Refractory Rickets: Identification of Novel PHEX Mutations in Indian Patients and a Literature Update

“…12 It is the predominant form of hypophosphatemic rickets with a prevalence of 1 in 20,000 and accounts for approximately 80% of the familial cases of hypophosphatemic rickets. 13 The PHEX gene is located on Xp22.1, is composed of 22 exons, and encodes a transmembrane endopeptidase that belongs to the type II integral membrane zinc-dependent endopeptidase. 14 The PHEX protein is predominantly expressed on the surface of bone and teeth during osteoblast differentiation and loss of its function results in defective mineralization of bone.…”

“…Only a few genetic studies on Indian patients are available. 13,45,[65][66][67][68][69] Our study at a tertiary care hospital, is mainly cases of refractory rickets that were referred for diagnosis and treatment. This study aimed at identifying mutations responsible for the pathogenesis of refractory rickets.…”

Delayed diagnosis and difficulties in the management of hypophosphatemicrickets in a 7-year-old girl – a case report

“…Najlepiej poznaną oraz najczęściej występującą w tej grupie krzywic jest krzywica hipofosfatemiczna związana z chromosomem X (XLH). Szacuje się, że schorzenie to dotyka 1 na 20 000 żywych urodzeń [1,10,12]. Niezależnie od rodzaju dziedziczenia leczenie polega na doustnej podaży fosforanów, preparatów wapnia oraz witaminy D lub jej analogów -kalcytriolu [5,13,14] lub alfakalcydolu [15].…”