Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

BACKGROUND: Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. METHODS: Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6-39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. RESULTS: Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%-100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%-95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. CONCLUSIONS: We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

Non-invasive prenatal diagnostic testing for ß-thalassaemia using cell-free fetal DNA and next generation sequencing.

OBJECTIVE: To develop an accurate non-invasive prenatal test using next generation sequencing (NGS) for HbE and the four most common ß-thalassaemia mutations found in South East Asia (namely -28A > G, CD17A > T, CD41/42(-TTCT) and IVS-II-654C > T). METHODS: Cell-free DNA was extracted from maternal plasma from 83 families where both parents were carriers of the HbE mutation or one of four common ß-thalassaemia mutations. Overlapping PCR amplicons covering each mutation were generated, pooled and sequenced using the Illumina MiSeq. Fastq files were analysed to detect inheritance of the paternal mutation. RESULTS: In two cases where the fathers were compound heterozygotes for HbE and -28A > G, the fetus was correctly diagnosed as having inherited one of the paternal mutations. In 35/85 cases, the paternal mutation was not detected, and in 50/85 cases, it was classified as inherited. Overall sensitivity for detection of paternal mutations was 100% (95% CI: 92.4-100%), and specificity was 92.1% (95% CI: 79.2-97.3%). CONCLUSION: We demonstrated that detection of paternal mutations using NGS can be readily achieved with high sensitivity and specificity, removing the need for an invasive test in 50% of pregnancies at risk of a thalassaemia in cases where the father and mother carry a different mutation. © 2014 John Wiley & Sons, Ltd.

Rapid prenatal diagnosis of common beta-thalassemia mutations in Southeast Asia using pyrosequencing.

OBJECTIVE: Current methods of prenatal diagnosis to detect beta-thalassemia are Sanger sequencing and reverse dot blot. These methods are time-consuming and can prolong assay turnaround time. We aim to develop a sensitive and rapid method to detect 27 beta-thalassemia mutations using pyrosequencing. METHOD: Pyrosequencing primer pairs and sequencing primers were designed to detect 27 most common beta-thalassemia mutations found in Singapore. Pyrosequencing was performed on 191 DNA samples with known beta-thalassemia mutations isolated from 143 peripheral blood and 48 prenatal samples (seven chorionic villus biopsies, 26 cultured amniocytes, 15 uncultured amniocytes). All mutations were validated with Sanger sequencing. RESULTS: Pyrosequencing identified 210 alleles with beta-thalassemia mutations and 82 alleles without mutations with 100% sensitivity (lower 95% confidence interval [CI], 97.8%) and 100% specificity (lower 95% CI, 94.4%). All pyrosequences were concordant with Sanger-based sequences. Pyrosequencing was able to detect DNA concentrations as low as 2 ng, obviating the need for cell culture in volume-restricted samples. Sample receipt-to-report assay turnaround times were 16 to 18 h (Sanger sequencing) and 4 to 6 h (pyrosequencing). CONCLUSION: Pyrosequencing is a rapid and sensitive method to detect common beta-thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time.

Targeting both rs12979860 and rs8099917 polymorphisms with a single-tube high-resolution melting assay for IL28b genotyping.

A rapid, duplex, high-resolution melting interleukin-28B gene (IL28B) genotyping assay, targeting both rs12979860 and rs8099917 polymorphisms, was developed and validated using 30 DNA samples from healthy volunteers. A linkage study on 300 healthy Singaporeans showed variable haplotypes. When the assay was applied to plasma DNA from 50 hepatitis C virus genotype-1 (HCV-1)-infected patients, five compound heterozygous types were detected.

Microsatellite markers within --SEA breakpoints for prenatal diagnosis of HbBarts hydrops fetalis.

BACKGROUND: We sought to develop a rapid prenatal diagnostic test for simultaneous detection of HbBarts hydrops fetalis and exclusion of maternal contamination. METHODS: We developed a multiplex quantitative fluorescent PCR (QF-PCR) test that detects the presence/ absence of 2 microsatellite markers (16PTEL05/16PTEL06) located within breakpoints of the Southeast Asia ((-SEA)) deletion. HbBarts hydrops fetalis ((-SEA/-SEA)) is diagnosed by absence of both markers, and maternal contamination of fetal DNA is excluded by absence of noninherited maternal alleles. Fetal and parental DNA samples from 50 families were analyzed in a blinded clinical validation study, and QF-PCR results were compared with their respective molecular genotypes. RESULTS: The multiplex QF-PCR results included correct diagnoses of HbBarts hydrops fetalis in 11 of the fetuses tested, correct verification as unaffected in 20 fetuses, and correct identification as either carriers (alphaalpha/(-SEA)) or unaffected homozygotes in 18. Misidentification as unaffected occurred for 1 carrier. Sensitivity for diagnosis of HbBarts hydrops fetalis was 100% [lower 95% confidence interval, 76.2%], and specificity was 100% (lower 95% confidence interval, 92.6%). None of the samples tested showed any traces of noninherited maternal alleles; thus false-positives because of maternal contamination were eliminated. CONCLUSIONS: In this QF-PCR method, detection of maternally and paternally inherited fetal alleles allowed diagnosis of the double-deletion syndrome, and the ability to differentiate between these alleles allowed simultaneous exclusion of maternal contamination of the fetal genetic material. This novel strategy using cell-free fetal DNA in maternal plasma could form the basis for noninvasive testing for HbBarts hydrops fetalis.