Non-invasive prenatal diagnosis (NIPD) of isolated cell-free DNA from maternal plasma has been applied to detect monogenic diseases in the fetus. Droplet digital PCR (ddPCR) is a sensitive and quantitative technique for NIPD. In the present study, the development and evaluation of ddPCR-based assays for common α and β-thalassemia variants amongst the Asian population was described; specifically, Southeast Asian (SEA) deletion, HbE, and 41/42 (-CTTT). SEA is caused by deletion of a 20 kb region surrounding the α-globin gene, whilst HbE and 41/42 (-CTTT) are caused by point mutations on the β-globin gene. Cell-free DNA samples from 46 singleton pregnant women who were carriers of these mutations were isolated and quantified using ddPCR with specially designed probes for each target allele. Allelic copy number calculation and likelihood ratio tests were used to classify fetal genotypes. Classification performances were evaluated against ground truth fetal genotypes obtained from conventional amniocentesis. Copy number variation analysis of SEA deletion accurately classified fetal genotypes in 20 out of 22 cases with an area under the receiver operating characteristic curve of 0.98 for detecting Hb Bart's hydrops fetalis. For HbE cases, 10 out of 16 samples were correctly classified, and three were inconclusive. For 41/42 (-CTTT) cases, 2 out of 8 were correctly classified, and four were inconclusive. The correct genotype was not rejected in any inconclusive case and may be resolved with additional ddPCR experiments. These results indicate that ddPCR-based analysis of maternal plasma can become an accurate and effective NIPD for SEA deletion α-(0) thalassemia. Although the performance of ddPCR on HbE and 41/42 (-CTTT) mutations were not sufficient for clinical application, these results may serve as a foundation for future works in this field.

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For decades, diverse plasmid vectors have been continuously developed for molecular cloning of DNA fragment in the bacterial host cell Escherichia coli. Even with deliberate performances in vector preparation, the cloning approaches still face inevitable background colonies, or false positive clones, that may be arisen from intact or self-ligated plasmid molecules. To assist in such problem, two plasmids, pBS2ndd and pBS3ndd, which resistant to ampicillin and kanamycin respectively, were developed in this study as more advantageous cloning vector. The plasmids carry ndd, a lethal gene from bacteriophage T4 coding for nucleoid disruption protein that binds to the host chromosome and progressively kill the cell. The deadly toxicity of Ndd inhibits host cells that obtain intact or ndd-religated vector from growing, which results in low background and dramatically reduces the effort for selection of recombinants. Moreover, their identical multiple cloning site was designed to support various cloning strategies. Digestion of plasmids with XcmI allows for in vitro T/A ligation, while with EcoRV permits blunt-end ligation, with capability of blue-white colony screening. In vivo homologous recombination cloning is also utilizable by amplification of insert fragments using primers containing homology arms and transformation into capable E. coli strains. To demonstrate their advantages, the plasmids were used to clone PCR product samples for DNA sequencing with low-background and versatile cloning strategies. Such rapid and cost-effective cloning procedures are also proposed here. Finally, the cloning for protein expression with blue-white selection was also possible using egfp as a model regulated by lac and T7 promoters on the plasmid or other build-in promoters with the insert.

Objective The aim of this study is to develop ddPCR based-assay for detecting alpha (0)-thalassemia (SEA) and beta-thalassemia (HbE and 41/42 (-CTTT) from cell-free fetal DNA (cffDNA) extracted form maternal plasma. Design Feasibility study using sample collected from prenatal clinic. Setting Thailand. Population 46 couples who were identified to be carriers of alpha or beta thalassemia. Method Cell-free DNA from 46 singleton pregnant women were isolated and quantified using ddPCR with specially designed probes for each target allele. Allelic copy number (CNV) calculation and likelihood ratio test were used to classify the most likely fetal genotypes. Classification performances were evaluated against ground truth fetal genotypes obtained from conventional amniocentesis. Main outcome measures Concordance with fetal genotyping results from invasive technique. Sensitivity and specificity of ddPCR-based assays. Results CNV analysis of SEA deletion accurately classify fetal genotypes in 20 out of 22 cases with an AUC of 0.98 (95% sensitivity and 91% specificity) for the prediction of Hb Bart's hydrops fetalis. Application of sequential probability ratio tests to detect HbE and 41/42 correctly classified 12 out of 24 cases (10 out of 16 HbE and 2 out of 8 41/42) and provided inconclusive for 7 cases. Conclusion We showed that ddPCR-based analysis of maternal plasma is an accurate and effective NIPD for SEA deletion. Although the performance of ddPCR-based assay on HbE and 41/42 mutations is still not high enough for clinical application, our work should serve as a good foundation for future works in this field. Hosted fileThalassemia_ddPCR_manuscript_BJOG_092720.pdf available at https://authorea.com/users/362445/ articles/483671-droplet-based-digital-pcr-for-non-invasive-prenatal-genetic-diagnosisfor-alpha-and-beta-thalassemia-a-feasibility-study