MOLECULAR PATHOLOGY: POTENTIAL BIOMARKERS FOR THE DETECTION OF DOWN SYNDROME PREGNANCIES

Abstract

Down Syndrome (DS), also called trisomy 21, is the most common non- lethal fetal aneuploidy that affects 1 in 800 live births. The disease appears mostly due to the existence of an extra copy of chromosome 21. In the UK, the screening of DS is offered to all pregnant women in the antena- tal care program to assess the risk of DS. If the risk is high the women are offered further testing that includes invasive sampling (amniocente- sis or chorionic villus sampling) to confirm the diagnosis. This project aimed to investigate the possibility of applying new methods to screen for DS which will give women at a medium risk of having a DS baby the chance to avoid unnecessary invasive interventions. These methods in- clude liquid chromatography mass spectrometry (LCMS) to identify pos- sible new biomarkers for DS pregnancies and investigate the potential use of PRSS56 and SPINK7 proteins as two new target biomarkers for DS. We will also use multiplex droplet digital PCR (ddPCR) using cell-free fetal DNA (cffDNA) to target different genes on chromosomes 21, 18, and 13 to screen for trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), respectively. PRSS56 and SPINK7 proteins are secreted proteins and found to be highly expressed in the DS fetus placenta. The level of these proteins was investi- gated using depleted plasma samples from DS pregnancies and compared to plasma samples from healthy individuals (controls). The plasma sam- ples were subjected to immunoprecipitation and Western blotting using monoclonal and polyclonal antibodies. PRSS56 was not detected in theplasma samples from DS pregnancies. SPINK7 was difficult to detect due to its small size and furthermore optimisation is needed. A group of 14 depleted plasma samples from DS and healthy control preg- nancies were tested by LCMS to evaluate the abundance of the proteins in each group. The differential analysis showed 47 proteins were abundant in DS pregnancies with more than 2-fold change compared to control sam- ples. Following the presence/absence analysis on the mass spectrometry dataset, the actin-related protein 2/3 complex subunit 2 (ARPC2) was de- tected in controls and absent in most of the DS pregnancies. ddPCR was investigated as a possible non-invasive prenatal testing (NIPT) platform to assess its ability and efficiency to analyse cffDNA. Samples were spiked by trisomy genomic DNA for T21, T18 and T13 and were tested to mimic the low fraction of cffDNA in the maternal blood samples. The T21 multiplexing assay targeted HSPA13 and VPS26C genes located on chromosome 21 against LEPROT and AGO1 genes located on on chro- mosome 1 as reference genes. The T21, T18 and T13 multiplex assay tar- geted VPS26C gene located on chromosome 21, BRCA2 gene located on chromosome 13 and LPIN2 gene located on chromosome 18 against AGO1 gene located on chromosome 1 as a reference. After the optimisation, 16 clusters were observed and each cluster was isolated with minimum rain- drops, where the threshold lines can be easily placed between clusters. In conclusion, finding new biomarkers for DS will improve the detection rate of screening assays, which will reduce the need for invasive interven- tions. Studying protein abundance in DS pregnancies and different path- ways will help us to find new biomarkers and understand their potential involvement in the severity of the disease. NIPT requires a rapid, sensi- tive and simple platform that is capable to test cffDNA efficiently. ddPCR has the potential to replace next-generation sequencing and microarrays because it is cheaper and easier to perform on a daily basis routine.