Investigation into Inter-alpha Trypsin Inhibitor Heavy Chain 4 (ITIH4) as a biomarker for prenatal screening of Down syndrome using maternal plasma samples

Abstract

Background: Down Syndrome (DS) effects 3 per 1000 births and is a significant cause of fetal loss. Currently screening tests detect ~85% of affected pregnancies, with a 5% false-positive rate, whilst diagnosis requires invasive tests such as amniocentesis which is associated with a ~1% risk of miscarriage. More effective screening methods, or Non Invasive Prenatal Diagnosis (NIPD) methods, are required to reduce the need for invasive diagnostic tests. Aim: This study aims to investigate inter-alpha trypsin inhibitor heavy chain 4 (ITIH4) as a potential biomarker in DS pregnancies. Method: Samples include 8 non-pregnant controls and 7 pregnant women known to be carrying a DS fetus. 1D Western Blot analysis was carried out on all plasma samples, and 2D Western blot (2-DE) was subsequently carried out on samples of interest. Real-time PCR was undertaken to determine the sex of the fetuses. Results: 125kDa ITIH4 was identified in all samples using Western Blot analysis. ITIH4 is cleaved by kallikrein into a 35kDa fragment and a 100kDa fragment which is rapidly cleaved to yield a 70kDa fragment. Results for DS samples KC05 and KC06 suggested only the 35kDa fragment was present, whilst all other samples demonstrated the presence of the full 125kDa ITIH4 protein, as well as the presence of the 35kDa fragment in some cases. DS samples KC01 and KC05 underwent 2D Western blot. Results for KC05 again only presented the 35kDa fragment, whilst KC01 presented both the fragment and full ITIH4 protein in contrast to the previous 1D Western Blot which had only demonstrated the presence of the full ITIH4 protein. All DS fetal genders were successfully identified. Conclusion: ITIH4 was successfully identified using the 1D and 2D Western Blot, in all samples. Reasons for the presence of different fragment of ITIH4 in different plasma samples remain unclear and require further investigation. Further research needs to be undertaken to compare euploid samples with known trisomy samples on a larger scale to identify whether ITIH4 could be an effective biomarker in DS screening.