Non‐invasive prenatal diagnosis of achondroplasia and thanatophoric dysplasia: next‐generation sequencing allows for a safer, more accurate, and comprehensive approach

Abstract

ObjectiveAccurate prenatal diagnosis of genetic conditions can be challenging and usually requires invasive testing. Here, we demonstrate the potential of next-generation sequencing (NGS) for the analysis of cell-free DNA in maternal blood to transform prenatal diagnosis of monogenic disorders.MethodsAnalysis of cell-free DNA using a PCR and restriction enzyme digest (PCR–RED) was compared with a novel NGS assay in pregnancies at risk of achondroplasia and thanatophoric dysplasia.ResultsPCR–RED was performed in 72 cases and was correct in 88.6%, inconclusive in 7% with one false negative. NGS was performed in 47 cases and was accurate in 96.2% with no inconclusives. Both approaches were used in 27 cases, with NGS giving the correct result in the two cases inconclusive with PCR–RED.ConclusionNGS provides an accurate, flexible approach to non-invasive prenatal diagnosis of de novo and paternally inherited mutations. It is more sensitive than PCR–RED and is ideal when screening a gene with multiple potential pathogenic mutations. These findings highlight the value of NGS in the development of non-invasive prenatal diagnosis for other monogenic disorders. © 2015 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.What's already known about this topic?Non-invasive prenatal diagnosis (NIPD) using PCR-based methods has been reported for the detection or exclusion of individual paternally inherited or de novo alleles in maternal plasma.What does this study add?NIPD using next generation sequencing provides an accurate, more sensitive approach which can be used to detect multiple mutations in a single assay and so is ideal when screening a gene with multiple potential pathogenic mutations. Next generation sequencing thus provides a flexible approach to non-invasive prenatal diagnosis ideal for use in a busy service laboratory.

Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends

“…Thus, it is clinically relevant to be able to screen for de novo mutations prenatally. Efforts in the noninvasive prenatal detection of fetal de novo mutations are limited to diseases associated with structural abnormalities detectable on ultrasound and where those diseases are known to be associated with hotspot de novo mutations (24 >90% of achondroplasia cases are caused by a de novo mutation in the FGFR3 (fibroblast growth factor receptor 3) gene with no prior familial history of skeletal dysplasia (25). A clinical suspicion for achondroplasia could, therefore, be followed by maternal plasma DNA analysis with the aim to specifically detect fetal FGFR3 mutations (26).…”

Extending non-invasive prenatal testing to non-invasive prenatal diagnosis

“…In the early stages of the technology, multiple assays were developed to detect inheritance of paternal genetic changes or de novo genetic changes for various different autosomal dominant conditions. The process started to become more streamlined in 2012 when NIPD for achondroplasia and thanatophoric dysplasia became approved for NHS use 4. NIPD has also been used in the context of recessive conditions—initially in order to detect whether the relevant paternal genetic change had been inherited or not (if it had, the couple would then be offered an invasive prenatal test to check for inheritance of the relevant maternal genetic change).…”

Noninvasive Prenatal Screening for Genetic Diseases Using Massively Parallel Sequencing of Maternal Plasma DNA

“…At the moment, these are used in clinical practice for the detection or exclusion of the paternally inherited in recessive conditions such as cystic fibrosis in which the parents carry different mutations (Hill et al 2015) or for the detection of dominantly inherited conditions in which the father carries the mutation or it arises de novo. A number of these panels have now been approved for use in the NHS by the UKGTN, including an FGFR3 panel for the diagnosis of achondroplasia, thanatophoric dysplasia and other FGFR3-related skeletal dysplasias (http://ukgtn.nhs.uk/ find-a-test/search-by-disorder-gene/test-service/ fgfr3-related-skeletal-dysplasias-panel-test-nipd-654/; Chitty et al 2015), an FGFR2 panel for Apert syndrome (http://ukgtn.nhs.uk/find-atest/search-by-disorder-gene/test-service/apertsyndrome-nipd-697/) and a panel covering the 10 most common cystic fibrosis mutations (http: //ukgtn.nhs.uk/find-a-test/search-by-disordergene/details/5062/). The sequencing approach offers flexibility, the ability to screen a case for multiple mutations at one time, and several cases can be multiplexed either when testing for the same condition or multiple conditions.…”