In 2014, CHECT and Fight for Sight co-funded a research project to improve the sensitivity of current genetic testing methods that all new families in the UK affected by retinoblastoma now undergo. Project leaders Dr Simon Ramsden and Dr Trevor Cole talk us through the aims and achievements of the research…

Retinoblastoma is caused by mutations in one of our genes – RB1 – and so we use genetic testing to advise on the risks to an affected child’s relatives.

Mutations can be either passed from parent to child or can arise spontaneously in the body. Once we have identified the cause by testing the RB1 gene in an affected child, we can offer testing to family members to see if they have a high or low risk of developing Rb. In this way, we can also reassure those families where Rb is non-genetic.

Most cases of Rb arise in a child with no family history of the disease and we first look for the causative mutation in the patient’s blood. However, in about 50% of these children we are not able to detect a mutation, which may limit our ability to offer accurate risk prediction to family members.

There are different reasons for not being able to identify a mutation in a blood sample and the most likely one is that it is simply not present. However, this is not always the case. For patients who have not inherited a mutation from a parent, it will have arisen spontaneously during their development – either in the embryo or very shortly after birth.

If it has arisen in organs other than the eye, then there is a risk that the mutation is present in a subset of the cells in the body (so called mosaic patients). In this situation brothers and sisters are at low risk – in fact near the general population risk – of developing Rb. However, when the affected child grows to have a family of their own they may be at risk of passing on the mutation through the egg or sperm if the sub-set of cells with the mutation (ie the mosaic portion) includes cells in the ovaries or testicles respectively.

Mosaic mutations can be difficult to identify using conventional methods as you need to sequence DNA from large numbers of cells simultaneously in order to get a signal strong enough to detect. With this approach low level signals can be lost in the background “noise”. However, new and exciting DNA sequencing technologies are opening up new possibilities in healthcare and this is one area that can benefit.

These new sequencing technologies (so-called Next Generation Sequencing or NGS) were first developed to enable the sequencing of entire genomes but they can have other applications. CHECT research funding has supported the introduction of NGS into routine RB1 testing to increase the sensitivity of mutation detection in mosaic patients and allow us to rule out more family members from being at risk of Rb.

While there are a wide variety of NGS technologies they all share one important feature, namely that they sequence individual DNA molecules rather than a mixed pool of molecules. In fact they sequence many millions of molecules individually (but simultaneously) and then use computer software to compile a profile of all individual DNA molecules. In this way low level signals are not lost against the background noise, but instead counted by the software.

Thanks to the support of CHECT, we can now identify mutations at much lower levels in the blood than was previously possible. We estimate that we can now identify mosaic mutations in approximately 10% of patients that would have previously tested negative, which means that we can provide accurate risk estimation to more family members than ever before.

This, in turn, will enable us to exclude more siblings, previously deemed to be at risk, from costly and distressing surveillance.

Funding research

CHECT is committed to supporting research which is a vital part of the fight against Rb. In 2015/2016 we supported eight ongoing research projects and funded three new ones.

We don’t receive any government funding and the money for this project was raised through the tremendous efforts of our members who took part in the Carrots Nightwalks 2012 and 2013. A huge thank you to all of you who made this important development in genetic testing possible.

About the authors

Dr Simon Ramsden is Consultant Clinical Scientist at Manchester Centre for Genomic Medicine.

Dr Trevor Cole is Consultant and Honorary Reader in Clinical and Cancer Genetics at Birmingham Women’s NHS Foundation Trust.

This article featured in the latest edition of our newsletter InFocus, out now. You can download a copy on our publications page.