Validation of a next-generation sequencing (NGS) panel to improve the diagnosis of X-linked hypophosphataemia (XLH) and other genetic disorders of renal phosphate wasting.

Susanne Thiele,Ralf Werner,Annika Stubbe,Wolfgang Hoeppner,Olaf Hiort

doi:10.1530/eje-20-0275

Susanne Thiele, Ralf Werner + Show 3 more

Open Access

https://doi.org/10.1530/eje-20-0275

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Journal: Acta Endocrinologica	Publication Date: Aug 14, 2020
Citations: 10	License type: CC BY 4.0

Affiliation: University of Lübeck, Bioglobe (Germany)

Abstract

BackgroundHypophosphataemic rickets (HR) comprise a clinically and genetically heterogeneous group of conditions, defined by renal-tubular phosphate wasting and consecutive loss of bone mineralisation. X-linked hypophosphataemia (XLH) is the most common form, caused by inactivating dominant mutations in PHEX, a gene encompassing 22 exons located at Xp22.1. XLH is treatable by anti-Fibroblast Growth Factor 23 antibody, while for other forms of HR such as therapy may not be indicated. Therefore, a genetic differentiation of HR is recommended.ObjectiveTo develop and validate a next-generation sequencing panel for HR with special focus on PHEX.Design and methodsWe designed an AmpliSeq gene panel for the IonTorrent PGM next-generation platform for PHEX and ten other HR-related genes. For validation of PHEX sequencing 50 DNA-samples from XLH-patients, in whom 42 different mutations in PHEX and 1 structural variation have been proven before, were blinded, anonymised and investigated with the NGS panel. In addition, we analyzed one known homozygous DMP1 mutation and two samples of HR-patients, where no pathogenic PHEX mutation had been detected by conventional sequencing.ResultsThe panel detected all 42 pathogenic missense/nonsense/splice-site/indel PHEX-mutations and in one the known homozygous DMP1 mutation. In the remaining two patients, we revealed a somatic mosaicism of a PHEX mutation in one; as well as two variations in DMP1 and a very rare compound heterozygous variation in ENPP1 in the second patient.ConclusionsThis developed NGS panel is a reliable tool with high sensitivity and specificity for the diagnosis of XLH and related forms of HR.

Highlights

X-linked hypophosphataemia (XLH) (OMIM#307800) is the most common genetic disorder of phosphate homeostasis characterized by renal phosphate wasting and hypophosphataemia
This sample was subsequently analyzed by MLPA and revealed a duplication of PHEX exon 12, confirming previous results (Supplementary Fig. 1)
Improvements in gene sequencing technologies in combination with rapidly declining costs have led to the development of a large amount of targeted next-generation sequencing (NGS) panels

Summary

Introduction

X-linked hypophosphataemia (XLH) (OMIM#307800) is the most common genetic disorder of phosphate homeostasis characterized by renal phosphate wasting and hypophosphataemia. It affects about one in 20 000 individuals [1] and follows an X-chromosomal dominant inheritance pattern. X-linked hypophosphataemia (XLH) is the most common form, caused by inactivating dominant mutations in PHEX, a gene encompassing 22 exons located at Xp22.1. Objective: To develop and validate a next-generation sequencing panel for HR with special focus on PHEX. We analyzed one known homozygous DMP1 mutation and two samples of HR-patients, where no pathogenic PHEX mutation had been detected by conventional sequencing. Results: The panel detected all 42 pathogenic missense/nonsense/splice-site/indel PHEX-mutations and in one the known homozygous DMP1 mutation.

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