Prenatal Diagnosis. Clinical Translation: Same day prenatal diagnosis
Rapid aneuploidy detection assay using quantitative fluorescence polymerase chain reaction (QF-PCR) of 19 microsatellite markers located on chromosomes 13, 18, 21, X and Y was validated in over one thousand samples from NUH. Available to patients since 2008. FlashFISH™ is a revolutionary technology that enables patients to receive the results of their amniocentesis test within three hours compared to conventional karyotyping of 8-21 days.
Chromosomes 13, 18, 21, X, and Y in each fetal cell are tagged with their respective fluorescent-labeled probes. This enables visualization and numeration of chromosomes under a fluorescence-enabled microscope. Developed and patented by National University of Singapore (NUS). INEX has exclusive license for FlashFISH™ technology which has been available to patients since 2011. Both tests are currently offered at the National University Hospital, (NUH) Singapore.
(A) In Utero Transplantation of human fetal MSC for Osteogenesis Imperfecta. A phase I/II open label clinical trial for IUMSCT in fetuses diagnosed with OI (multiple fractures on prenatal US imaging) commenced in 2010. This occurred on the basis of convincing evidence from preclinical models, and after the first treatment at the Karolinska Institute produced good evidence of safety and efficacy; to date we have performed the second and third cases globally, and the first two cases in Asia. Outcomes have been published and clinical surveillance is ongoing.
(B) Haemopoietic Stem Cells for In Utero Transplantation for major Haemoglobinopathies. Being the most common genetic disease in South East Asia, the major thalassaemias α and β represent a significant disease burden, and allogeneic IUHSCT presents an opportunity to prevent lethal or chronic morbidity. Our team’s strength has been our preclinical models, a murine thalassaemia model and a non-human primate model for study of long-term outcomes. Our original work and international collaborations have resulted being in a prime position to become co-trialists in upcoming clinical trials of IUHSCT for α (lead institute UCSF) and β thalassaemia (lead institute CHOP).
Whole, nucleated fetal cells (FNRBC), which are found in the maternal blood stream are considered promising candidates for detecting chromosomal abnormalities, as they can be cultured and contain the entire fetal genome. With the expansion and improvement in next generation sequencing platforms it is currently possible to map the entire fetal genome thereby identifying life threatening or crippling defects and disorders. Early identification could allow early treatment, even in utero, when relevant. In order to enable next generation sequencing of fetal cells, whole and viable cells must be gathered. To date this is still done invasively, via CVS or amniocentesis.
Circulating fetal cells are a promising source for non-invasive detection of aneuploidies as well as for providing complete genetic information of the fetus. These cells are very rare in the maternal blood stream.
We have developed a novel strategy, which consistently enriches FNRBCs from maternal blood and devised a technique to manually pick FNRBCs using a micromanipulator, based on the identification by size and fluorescent staining specific to FNRBCs.
Extensive characterization the FNRBC has enabled the development of a fetal erythroblast in-vitro culture protocol, which prevents their enucleation. This is crucial in maintaning the presence of the nucleus for downstream molecular diagnosis.
In our paper published in Prenatal Diagnosis (Hua & Barrett et al., 2014), we showed the feasibility of whole genome amplification (WGA) of DNA from single FNRBCs, followed by massively parallel sequencing, to detect trisomies 21, 18, and 15. This shows potential utility for definitive non-invasive prenatal diagnosis. Correct diagnoses were made from ten single cells isolated from villi from two cases of trisomy 21 (one case from a single cell and one from two cells), two cases of trisomy 18 (two cells each), and a case of trisomy 15 (three cells).
Cell-free Fetal DNA
Cell-free fetal DNA (cffDNA, derived from the trophoblast layer of the placenta, circulates in maternal blood, and is detectable from five week’s gestation onwards. Non-invasive prenatal testing for aneuploidy is widely available commercially, and is slowly being implemented by a number of national health services. In addition, tests are being developed for non-invasive prenatal diagnosis (NIPD) of monogenic disorders.
cffDNA constitutes 10-15% of a cfDNA sample, the remainder being maternal in origin. Due to the high maternal background, it can be difficult to identify maternally inherited fetal alleles; detection of paternal alleles is more straightforward using sensitive metods such as digital PCR and next generation sequencing (NGS). We have published a method using targeted NGS for detection of paternal inheritance of the five most common b-thalassemia mutations in couples carrying different mutations (Li & Barrett et al., 2014). If the paternal allele is not detected, the baby will either be a carrier (if the maternal allele has been inherited) or have a normal genotype, and so no further testing is required. When implemented into clinical practice, this test will reduce the need for invasive testing in carrier couples with different mutations by 50%. We are currently working on methods to detect inheritance of the maternal allele in couples with b-thalassaemia using relative mutation dosage (RMD) which, if successful, will remove the requirement for invasive testing completely.
RMD works by detection of the over-representation of a mutant allele, and the degree of over-representation is affected by the fetal fraction of the cfDNA sample. This is easy to measure in male samples, using Y chromosome sequences which are not present in the mother’s DNA. However, there is no universal fetal marker, simple methods for quantification of fetal fraction for RMD in female cases is much less readily achieved. We have developed a panel of informative insertion/deletion (indel) polymorphisms that can be used to detect paternally inherited indels, allowing quantification of fetal fraction in all pregnancies.
Using targeted NGS methods, we have demonstrated that we can detect paternal inheritance of the Hb Constant Spring and Hb Quong Sze point mutations that are commonly implicated in a-thalassaemia in South East Asia. However, the majority of a-thalassaemia is caused by inheritance of large deletions covering the a-globin genes. We are developing NIPD to detect these large deletions.