Forensic pregnancy diagnostics with placental mRNA markers

Current methods for pregnancy diagnostics are based on immunodetection of pregnancy-specific proteins and in a forensic context suffer from sensitivity and specificity issues. Here, we applied reverse transcriptase polymerase chain reaction (RT-PCR) technology to 11 genes previously reported with placental mRNA circulating in maternal blood. We found two genes, hPL and βhCG, with pregnancy-specific expression in whole blood samples. RT-PCR detection of hPL was positive in all samples tested throughout the pregnancy, whereas βhCG was detectable until half of the second trimester but not at later gestation ages. For hPL, in vitro stability of the transcript was demonstrated until 2 months of age, and the hPL-specific RT-PCR assay applied was highly sensitive with reliable detection from down to 0.25 cm2 dried bloodstain. We therefore suggest hPL-specific RT-PCR as a new molecular tool for forensic pregnancy diagnostics from dried blood stains. Moreover, our results indicate that the time-wise reverse expression of hPL and βhCG during pregnancy may allow an RT-PCR-based estimation of the gestational age from blood stains, adding to the value of forensic pregnancy diagnosis for crime scene investigations

Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study.

To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling. Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples. Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands. 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection. Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%. Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.published_or_final_versio

Comprehensive multiparameter genetic analysis improves circulating tumor DNA detection in head and neck cancer patients

Introduction: Tumor-specific genetic aberrations in cell-free DNA (cfDNA) from plasma are promising biomarkers for diagnosis of recurrent head and neck squamous cell carcinoma (HNSCC). However, the sensitivity when using somatic mutations only in cfDNA is suboptimal. Here, we combined detection of copy number aberrations (CNAs), human papillomavirus (HPV) DNA and somatic mutations in a single sequencing workflow. Methods: Pretreatment plasmas of 40 patients and 20 non-cancer controls were used for analysis. Plasma DNA underwent low-coverage whole genome sequencing (lcWGS) to detect both CNAs and HPV-DNA, and deep sequencing to detect mutations in 12 frequently altered cancer driver genes in HNSCC using the same sequencing library. A specific analysis pipeline line was developed for data mining. The corresponding tumors were analyzed using slightly adapted protocols. Results: Using the developed method, somatic mutations and CNAs were detected in plasma DNA of HNSCC patients in 67% and 52%, respectively. HPV-DNA in plasma was detected in 100% of patients with HPV-positive tumors, and not in plasma of patients with HPV-negative tumors or non-cancer controls. Combined analysis increased the detection rate of tumor DNA in plasma to 78%. The detection rate was significantly associated with the stage of disease of the tumor. Neither HPV status nor location of the primary tumor influenced detection of CNAs or somatic mutations in plasma. Conclusions: This study demonstrates that the combined analysis of CNAs, HPV and somatic mutations in plasma of HNSCC patients is feasible and contributes to a higher sensitivity of the assay compared to single modality analyses

Maternal segregation of the Dutch preeclampsia locus at 10q22 with a new member of the winged helix gene family

© 2005 Nature Publishing GroupPreeclampsia is a pregnancy-associated disease with maternal symptoms but placental origin. Epigenetic inheritance is involved in some populations. By sequence analysis of 17 genes in the 10q22 region with maternal effects, we narrowed the minimal critical region linked with preeclampsia in the Netherlands to 444 kb. All but one gene in this region, which lies within a female-specific recombination hotspot, encode DNA- or RNA-binding proteins. One gene, STOX1 (also called C10orf24), contained five different missense mutations, identical between affected sisters, cosegregating with the preeclamptic phenotype and following matrilineal inheritance. Four STOX1 transcripts are expressed in early placenta, including invasive extravillus trophoblast, generating three different isoforms. All contain a winged helix domain related to the forkhead (FOX) family. The largest STOX1 isoform has exclusive nuclear or cytoplasmic expression, indicating activation and inactivation, respectively, of the PI3K-Akt-FOX pathway. Because all 38 FOX proteins and all 8 STOX1 homologs have either tyrosine or phenylalanine at position 153, the predominant Y153H variation is highly mutagenic by conservation criteria but subject to incomplete penetrance. STOX1 is a candidate for preeclampsia controlling polyploidization of extravillus trophoblast.Marie van Dijk, Joyce Mulders, Ankie Poutsma, Andrea A M Könst, Augusta M A Lachmeijer, Gustaaf A Dekker, Marinus A Blankenstein and Cees B M Oudejan