A team of researchers from Hong Kong have developed a proof-of-concept RNA-sequencing approach to analyse amniotic fluid to diagnose rare disease. The work, published this week in npj Genomic Medicine, discusses the benefits of RNA-sequencing for the diagnosis of genetic disorders and its potential use in prenatal testing.
A vital field
The vast majority of rare congenital diseases have a genetic component. Early diagnosis of these disorders (and an understanding of the causes) is vital for both treatment and future family planning. Current diagnostic approaches include pre-natal screening methods, such as ultrasounds and analysis of placental cells (chorionic villus sampling), or post-natal blood tests.
Amniocentesis is an alternative to chorionic villus sampling that sees cells taken directly from the amniotic fluid in the womb. Amniocentesis is typically offered when a fetus is thought to be at a high risk of suffering from a genetic disorder; for example, if there is a family history or an abnormal ultrasound result. Amniotic fluid cells can be analysed to detect any variants of concern – not only during gestation, but also postnatally, if stored correctly.
Currently, traditional DNA sequencing methods can be used to detect fetal abnormalities. However, these methods have drawbacks. Results from these techniques cannot typically be used to assign pathogenicity to variants of uncertain significance. To combat this, RNA-sequencing is now being considered as a standard diagnostic tool for genetic disorders. The gene expression data from RNA-sequencing experiments can be used to interpret variants of uncertain significance, aiding in our understanding of their function. However, there is little data so far on the use of RNA-sequencing to analyse amniotic fluid cells for prenatal diagnosis.
Skin fibroblasts can be used for RNA-sequencing and generally yield good results, typically much better than equivalent results from whole blood cells. However, obtaining skin fibroblasts is an invasive procedure. Amniotic fluid has a similar embryonic origin to skin fibroblasts, which led Brian Chung Hon-Yin and his team to hypothesise that amniotic cells may also be amenable to RNA-sequencing analysis. Samples of amniotic fluid from 52 fetuses in the second trimester were analysed – 48 were healthy controls and 4 were samples from fetuses already diagnosed with a genetic disorder. Skin fibroblast and whole blood cell data was obtained from the GTEx server.
The first step in determining the potential of amniotic fluid in diagnostics is to determine how well genes are expressed in these samples. As hypothesised, gene expression was very similar to that in skin fibroblasts and much better and less variable than in blood (Figure 1). Many genes that have been previously implicated in rare disease could be detected at high levels in both amniotic fluid and skin fibroblasts. This result implied that amniotic fluid is in fact suitable for further analysis.
Figure 1: PCA plot showing the grouping of amniotic fluid cells, skin fibroblasts and whole blood cells. Each dot represents a different sample. Amniotic fluid and skin fibroblast cells group closely together, whilst blood cells exhibit significant differential gene expression and more variance. Taken from Lee et al. 2023.
To validate the usefulness of amniotic fluid in diagnostics, samples from fetuses previously identified as having rare diseases were subject to RNA-sequencing. The results matched up with the previous diagnoses that were obtained through more traditional genome sequencing, and also provided more information on the precise underlying disease mechanisms. For example, in one fetus a splicing variant was identified that impacted expression of the SOX gene. Amniotic fluid was particularly useful in this case, as the SOX gene is only expressed during embryonic development as such cannot be found in other cell types.
A new era
Efficient diagnosis of rare diseases in fetuses and infants is a key topic in the field of genomic medicine. The results of the study clearly show the benefit of using not only RNA-sequencing in diagnostics but also of analysing less conventional cell types such as amniotic fluid. The ability to assign function to variants of uncertain significance is also a key consideration, as this knowledge can be used not only to inform potential precision treatment but also in future pregnancy planning.
Obtaining these cells via amniocentesis is typically harmless and relatively easy, and the at the ease at which the cells can be stored provides potential for further analysis postnatally. This highlights the feasibility of the technique and could be a huge step forward in the rapid diagnosis of disease in newborns. The authors stated that the approach “may lead to a new era.”