Non-Invasive Prenatal Diagnosis of Retinoblastoma Inheritance by Combined Targeted Sequencing Strategies.

Chipo Mashayamombe-Wolfgarten,Simon Ramsden,Trevor Cole,Elizabeth Young,Michael Parks,Samantha Court,Sue Carless,Joshua Bott,Maureen McCalla,Samuel Clokie,Benjamin Bowns,David Goudie,Stephanie Allen,Amy Gerrish

doi:10.3390/jcm9113517

Abstract

Retinoblastoma, the most common childhood eye cancer, presents in two forms: heritable or sporadic. Heritable retinoblastoma is caused by a germline mutation in the RB1 gene. Early diagnosis of children at risk of inheriting an RB1 mutation is crucial to achieve optimal clinical outcome. Currently, the majority of genetic testing is performed on newborns, which has multiple disadvantages for both families and the healthcare system. We have developed a non-invasive prenatal diagnosis (NIPD) service for retinoblastoma, available from 8 weeks’ gestation, which uses a combination of massively parallel sequencing (MPS) techniques, dependent on the inheritance model. Detection of paternal or suspected de novo RB1 variants is achieved through amplicon-based MPS. NIPD of a fetus at risk of maternal inheritance is performed using capture-based targeted sequencing and relative haplotype dosage analysis. In addition, we show proof of principle of how capture-based sequencing can be used for de novo variants unsuitable for amplicon-based testing. In total, we report the NIPD of 15 pregnancies, results of which show 100% concordance with all postnatal testing performed at the time of publication (n = 12) with remaining pregnancies ongoing. NIPD of retinoblastoma therefore offers a viable alternative to newborn genetic testing.

Highlights

Retinoblastoma (Rb) is the most common childhood eye cancer with an incidence of ~1:15,000–1:20,000 live births
Over 99% of retinoblastomas are due to bi-allelic inactivation in the RB1 gene, caused by either two somatic mutations or an initial germline mutation followed by a subsequent somatic hit as described in Knudson’s two-hit hypothesis [2]
We previously developed non-invasive prenatal diagnosis (NIPD) for X-linked [11] and autosomal recessive disorders [12] which allows both maternal and paternal inheritance to be determined by relative haplotype dosage (RHDO) analysis

Summary

Introduction

Retinoblastoma (Rb) is the most common childhood eye cancer with an incidence of ~1:15,000–1:20,000 live births (reviewed in [1]). We previously developed NIPD for X-linked [11] and autosomal recessive disorders [12] which allows both maternal and paternal inheritance to be determined by relative haplotype dosage (RHDO) analysis This method overcomes the difficulty in detecting maternal variants and, as it uses linkage-based approach, does not require test development for family-specific variants, some of which may be unsuitable for direct detection. These tests are part of our routine clinical service in the UK, providing non-invasive prenatal diagnosis for pregnancies at risk of cystic fibrosis (CF), spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), with over 150 pregnancies tested since September 2016 [7]. We show how capture-based targeted MPS can be adapted for the direct detection of larger paternal or likely de novo mutations, which would not be possible using an amplicon-based test

Patient Samples

Sample Processing and DNA Extraction

Amplicon-Based MPS

Bioinformatic Analysis

Capture-Based Targeted MPS

Relative Haplotype Dosage Analysis

Result

Haplotype Analysis of Maternally Inherited Alleles

NIPD of 2 Mb RB1 De Novo Deletion

Discussion

Findings

PHE Publications Gateway Number