Noninvasive Prenatal Diagnosis of SEA-Thalassemia by Combining 1000 Genomes Database and Relative Haplotype Dosage.

To explore a noninvasive method for diagnosis of SEA-thalassemia and to investigate whether the regional factors affect the accuracy of this method. The method involved using a public database and bioinformatics software to construct parental haplotypes for proband and predicting fetal genotypes using relative haplotype dosage. We screened and downloaded sequencing data of couples who were both SEA-thalassemia carriers from the China National Genebank public data platform, and matched the sequencing data format with that of the reference panel using Ubuntu system tools. We then used Beagle software to construct parental haplotypes, predicted fetal haplotypes by relative haplotype dosage. Finally, we used Hidden Markov Model and Viterbi algorithm to determine fetal pathogenic haplotypes. All noninvasive fetal genotype diagnosis results were compared with gold standard gap-PCR electrophoresis results. Our method was successful in diagnosing 13 families with SEA-thalassemia carriers. The best diagnostic results were obtained when Southern Chinese Han was used as the reference panel, and 10 families showed full agreement between our noninvasive diagnostic results and the gap-PCR electrophoresis results. The accuracy of our method was higher when using a Chinese Han as the reference panel for haplotype construction in the Southern Chinese Han region as opposed to Beijing Chinese region. The combined use of public databases and relative haplotype dosage for diagnosing SEA-thalassemia is a feasible approach. Our method produces the best noninvasive diagnostic results when the test samples and population reference panel are closely matched in both ethnicity and geography. When constructing parental haplotypes with our method, it is important to consider the effect of region in addition to population background alone.

First-trimester noninvasive prenatal diagnosis of seven facioscapulohumeral muscular dystrophy type 1 families using SNP-based amplicon sequencing: An earlier, rapid and safer way.

The study is to explore the feasibility and value of SNP-based noninvasive prenatal diagnosis (NIPD) for facioscapulohumeral muscular dystrophy type 1 (FSHD1) in early pregnancy weeks. We prospectively collected seven FSHD1 families, with an average gestational age of 8+6. Among these seven couples, there were three affected FSHD1 mothers and four affected fathers. A multiplex-PCR panel comprising 402 amplicons was designed to selective enrich for highly heterozygous SNPs upstream of the DUX4 gene. Risk haplotype was constructed based on familial linkage analysis. Fetal genotypes were accurately inferred through relative haplotype dosage analysis using Bayes Factor. All tests were successfully completed in a single attempt, and no recombination events were detected. NIPD results were provided within a week, which is 4 weeks earlier than karyomapping and 7 weeks earlier than Bionano single-molecule optical mapping (BOM). Ultimately, five FSHD1 fetuses and two normal fetuses were successfully identified, with a 100% concordance rate with karyomapping and BOM. Therefore, SNP-based NIPD for FSHD1 was demonstrated to be feasible and accurate in early weeks of gestation, although the risk of recombination events cannot be completely eliminated. In the future, testing of more cases is still necessary to fully determine the clinical utility.

Exploring factors impacting haplotype-based noninvasive prenatal diagnosis for single-gene recessive disorders.

Haplotype-based noninvasive prenatal diagnosis (NIPD) is applicable for various recessive single-gene disorders in proband families. However, a comprehensive exploration of critical factors influencing the assay performance, such as fetal fraction, informative single nucleotide polymorphism (SNP) count, and recombination events, has yet to be performed. It is critical to identify key factors affecting NIPD performance, including its accuracy and success rate, and their impact on clinical diagnostics to guide clinical practice. We conducted a prospective study, recruiting 219 proband families with singleton pregnancies at risk for eight recessive single-gene disorders (Duchenne muscular dystrophy, spinal muscular atrophy, phenylketonuria, methylmalonic acidemia, hemophilia A, hemophilia B, non-syndromic hearing loss, and congenital adrenal hyperplasia) at 7-14 weeks of gestation. Haplotype-based NIPD was performed by evaluating the relative haplotype dosage (RHDO) in maternal circulation, and the results were validated via invasive prenatal diagnosis or newborn follow-ups. Among the 219 families, the median gestational age at first blood draw was 8+5 weeks. Initial testing succeeded for 190 families and failed for 29 due to low fetal fraction (16), insufficient informative SNPs (9), and homologous recombination near pathogenic variation (4). Among low fetal fraction families, successful testing was achieved for 11 cases after a redraw, while 5 remained inconclusive. Test failures linked to insufficient informative SNPs correlated with linkage disequilibrium near the genes, with F8 and MMUT exhibiting the highest associated failure rates (14.3% and 25%, respectively). Homologous recombination was relatively frequent around the DMD and SMN1 genes (8.8% and 4.8%, respectively) but led to detection failure in only 44.4% (4/9) of such cases. All NIPD results from the 201 successful families were consistent with invasive diagnostic findings or newborn follow-up. Fetal fraction, informative SNPs count, and homologous recombination are pivotal to NIPD performance. Redrawing blood effectively improves the success rate for low fetal fraction samples. However, informative SNPs count and homologous recombination rates vary significantly across genes, necessitating careful consideration in clinical practice. We have designed an in silico method based on linkage disequilibrium data to predict the number of informative SNPs. This can identify genomic regions where there might be an insufficient number of SNPs, thereby guiding panel design. With these factors properly accounted for, NIPD is highly accurate and reliable.

Fetal RHD Screening in RH1 Negative Pregnant Women: Experience in Switzerland.

RH1 incompatibility between mother and fetus can cause hemolytic disease of the fetus and newborn. In Switzerland, fetal RHD genotyping from maternal blood has been recommended from gestational age 18 onwards since the year 2020. This facilitates tailored administration of RH immunoglobulin (RHIG) only to RH1 negative women carrying a RH1 positive fetus. Data from 30 months of noninvasive fetal RHD screening is presented. Cell-free DNA was extracted from 7192 plasma samples using a commercial kit, followed by an in-house qPCR to detect RHD exons 5 and 7, in addition to an amplification control. Valid results were obtained from 7072 samples, with 4515 (64%) fetuses typed RHD positive and 2556 (36%) fetuses being RHD negative. A total of 120 samples led to inconclusive results due to the presence of maternal or fetal RHD variants (46%), followed by women being serologically RH1 positive (37%), and technical issues (17%). One sample was typed false positive, possibly due to contamination. No false negative results were observed. We show that unnecessary administration of RHIG can be avoided for more than one third of RH1 negative pregnant women in Switzerland. This reduces the risks of exposure to a blood-derived product and conserves this limited resource to women in actual need.

Noninvasive prenatal diagnosis of Mendelian disorders for consanguineous couples by relative genotype dosage.

Noninvasive prenatal diagnosis relies on the presence in maternal blood of circulating cell-free fetal DNA released by apoptotic trophoblast cells. Widely used for aneuploidy screening, it can also be applied to monogenic diseases (NIPD-M) in case of known parental mutations. Due to the confounding effect of maternal DNA, detection of maternal or biparental mutations requires relative haplotype dosage (RHDO), a method relying on the presence of SNPs that are heterozygous in one parent and homozygous in the other. Unavoidably, there is a risk of test failure by lack of such informative SNPs, an event particularly likely for consanguineous couples who often share common haplotypes in regions of identity-by-descent. Here we present a novel approach, relative genotype dosage (RGDO) that bypasses this predicament by directly assessing fetal genotype with SNPs that are heterozygous in both parents (frequent in regions of identity-by-descent). We show that RGDO is as sensitive as RHDO and that it performs well over a large range of fetal fractions and DNA amounts, thereby opening NIPD-M to most consanguineous couples. We also report examples of couples, consanguineous or not, where combining RGDO and RHDO allowed a diagnosis that would not have been possible with only one approach.

Terminating Routine Cord Blood RhD Typing of the Newborns to Guide Postnatal Anti-D Immunoglobulin Prophylaxis Based on the Results of Fetal RHD Genotyping.

INTRODUCTION: Targeted routine antenatal prophylaxis with anti-D immunoglobulin (Ig) only to RhD-negative pregnant women who carry RhD-positive fetuses (determined by fetal RHD genotyping) has reduced D-alloimmunization significantly when administered in addition to postnatal prophylaxis. Achieving high analysis sensitivity and few false-negative fetal RHD results will make RhD typing of the newborn redundant. Postnatal prophylaxis can then be given based on the result of fetal RHD genotyping. Terminating routine RhD typing of the newborns in cord blood will streamline maternity care. Accordingly, we compared the results of fetal RHD genotyping with RhD typing of the newborns. METHODS: Fetal RHD genotyping was performed, and antenatal anti-D Ig was administered at gestational week 24 and 28, respectively. Data for 2017-2020 are reported. RESULTS: Ten laboratories reported 18,536 fetal RHD genotypings, and 16,378 RhD typing results of newborns. We found 46 false-positive (0.28%) and seven false-negative (0.04%) results. Sensitivity of the assays was 99.93%, while specificity was 99.24%. CONCLUSION: Few false-negative results support the good analysis quality of fetal RHD genotyping. Routine cord blood RhD typing will therefore be discontinued nationwide and postnatal anti-D Ig will now be given based on the result of fetal RHD genotyping.

Research Progress of Cell-Free Fetal DNA in Non-Invasive Prenatal Diagnosis of Thalassemia.

Thalassemia is a genetic disease that seriously affects the health of the fetus. At present, invasive prenatal diagnosis is the main method of thalassemia screening, but invasive prenatal diagnosis has the risk of fetal abortion. The discovery of cell-free fetal DNA (cffDNA) in the peripheral blood of pregnant women provides the possibility for non-invasive prenatal diagnosis (NIPD). Rapid and efficient capture of mutational information on cffDNA in maternal plasma can help prevent the birth of children with thalassemia major. Currently, strategies for cffDNA-based NIPD of thalassemia include the detection of paternal mutations in maternal plasma, detection of a proportion of wild and mutant alleles in maternal plasma, linkage disequilibrium single nucleotide polymorphism (SNP) based on pedigree probands, and prediction of fetal genotypes by bioinformatics combined with population information. Therefore, this paper will focus on the above aspects, in order to provide an essential reference to the prevention and treatment of thalassemia.

Haplotype-Assisted Noninvasive Prenatal Diagnosis of Genetic Diseases by Massively Parallel Sequencing of Maternal Plasma Cell-Free DNA.

Early prenatal diagnosis of genetic diseases allows for timely intervention or prevention of  the diseases in newborns. Conventional prenatal diagnosis of most genetic diseases relies on testing fetal DNA obtained by invasive procedures such as amniocentesis or chorionic villus sampling, which are associated with small risks of fetal loss. Maternal circulating blood contains cell-free DNA (cfDNA) from the fetal genome and can thus be used to noninvasively detect fetal genetic diseases such as chromosomal abnormalities, copy number variants, and single gene diseases. However, due to the presence of a high level of maternal cfDNA in the maternal blood stream, a relative haplotype dosage (RHDO) analysis is required to detect the mutant loci in the fetal genome when performing noninvasive prenatal diagnosis (NIPD) by massively parallel sequencing (MPS) of cfDNA. In this chapter, we describe a protocol utilizing the RHDO strategy for NIPD of any gene of interest associating with single gene diseases.

Noninvasive prenatal diagnosis targeting fetal nucleated red blood cells.

Noninvasive prenatal diagnosis (NIPD) aims to detect fetal-related genetic disorders before birth by detecting markers in the peripheral blood of pregnant women, holding the potential in reducing the risk of fetal birth defects. Fetal-nucleated red blood cells (fNRBCs) can be used as biomarkers for NIPD, given their remarkable nature of carrying the entire genetic information of the fetus. Here, we review recent advances in NIPD technologies based on the isolation and analysis of fNRBCs. Conventional cell separation methods rely primarily on physical properties and surface antigens of fNRBCs, such as density gradient centrifugation, fluorescence-activated cell sorting, and magnetic-activated cell sorting. Due to the limitations of sensitivity and purity in Conventional methods, separation techniques based on micro-/nanomaterials have been developed as novel methods for isolating and enriching fNRBCs. We also discuss emerging methods based on microfluidic chips and nanostructured substrates for static and dynamic isolation of fNRBCs. Additionally, we introduce the identification techniques of fNRBCs and address the potential clinical diagnostic values of fNRBCs. Finally, we highlight the challenges and the future directions of fNRBCs as treatment guidelines in NIPD.

A feasibility study of noninvasive prenatal diagnosis in facioscapulohumeral muscular dystrophy type 1 in a Chinese family.

Objective: To demonstrate the feasibility of haplotype-based noninvasive prenatal diagnosis of Facioscapulohumeral Muscular Dystrophy type 1 (FSHD1). Methods: Bionano optical mapping was used to identify the D4Z4 structural variation of the genomic DNA sample from the proband affected with FSHD1. In addition, based on the technique of next generation sequencing, the pathogenic haplotype was determined by using trio strategy through genotyping his parents, and also fetal inheritance of paternal haplotypes was then deduced using the Hidden Markov Model. Results: Bionano optical mapping analysis revealed that the proband has only three D4Z4 repeats left in the 4q35 chromosomal region and a disease-permitting 4qA haplotype. The other normal allele of the proband contains 29 D4Z4 repeats and also a 4qA haplotype. The noninvasive cell-free fetal DNA (cffDNA)-based haplotype analysis suggested that the fetus inherited the pathogenic allele from his father and thus was predicted to be affected by FSHD1. In addition, Bionano optical mapping also demonstrated the presence of the pathogenic allele in the fetus by interrogating the genomic DNA from the amniotic fluid cells. Conclusion: Our study showed the cffDNA-based haplotyping was feasible for the noninvasive prenatal diagnosis of FSHD1, which is able to provide earlier testing results with a lower risk of miscarriage and infection than invasive techniques.

Noninvasive Prenatal Diagnosis of Fetal RHD Status Using Cell-free Fetal DNA in Maternal Plasma.

Background: The main cause of hemolytic disease of the fetus and newborn (HDFN) is the incompatibility of the RHD antigen between mother and fetus. Following the discovery of cell-free fetal DNA (cffDNA), noninvasive fetal RHD genotyping also became possible, which will help in the better management of immunized RHD negative mothers and in the targeted prenatal injection of Rho(D) immune globulin (RhIG). The objective of this study was to establish a reliable method with high accuracy to determine the fetal RHD genotype. Methods: The project was a prospective observational cohort study. After cell-free DNA (cfDNA) extraction from maternal plasma, fetal RHD genotyping was performed by duplex real-time polymerase chain reaction (PCR) and exons 5, 7, and 10 of the RHD gene were examined. SRY and RASSF1A genes were used as internal controls to confirm the presence of cffDNA in maternal plasma. Results: Out of 40 samples, 33 were RhD positive heterozygous mothers and 7 cases were RHD negative. In three cases where both the fetal RHD and SRY genotypes were negative, RASSF1A was amplified in cell-free DNA sample treated with the BstUI enzyme, and the presence of cffDNA was confirmed. Conclusion: The findings reveal that the strategy used in this study is reliable and it is possible to determine the fetal RHD status with high accuracy. The strategy can help targeted injection of RhIG and prevent unnecessary injection in RhD negative mothers who carry an RhD negative fetus.

Noninvasive isolation of transcervical trophoblast cells for fetal identification.

OBJECTIVE: To identify trophoblastic cells retrieved from the cervix at a gestational age (GA) of 5-9 weeks by a noninvasive modality in fetuses. METHOD: Transcervical cells (TCCs) were noninvasively extracted by a cytobrush using the Papanicolaou sampling method. TCCs were immunostained with antihuman leukocyte antigen (HLA)-G and anticytokeratin (CK)-7 antibodies to identify trophoblastic cells. Maternal finger blood, gestational sacs, and 20 trophoblastic cells collected by a laser-guided microscopic single-cell capture system were examined and compared by short tandem repeat (STR) genotyping. RESULTS: Forty-nine pregnant women with GA of 5-9 weeks and six nonpregnant healthy women were included in the study. Trophoblastic cells were identified in 37 (75.5%) TCC samples, among which 34 (69.4%) were eligible for STR genotyping analysis. No trophoblastic cells were identified in nonpregnant healthy women. The STR genotyping analyses revealed 24 female and 10 male fetuses. TCC trophoblastic cells exhibited the same STR profiles as gestational sac and maternal blood in all samples, which indicated that the TCC trophoblastic cells originated from fetuses. CONCLUSION: This primary study validated that trophoblastic cells from TCCs at GA 5-9 weeks originated from the fetus. Further studies are needed to verify whether this method can be used for early noninvasive prenatal diagnosis and paternity testing.

Noninvasive prenatal diagnosis based on cell-free DNA for tuberous sclerosis: A pilot study.

BACKGROUND: Noninvasive prenatal diagnosis (NIPD) based on cell-free DNA (cfDNA) has been introduced into the clinical application for some monogenic disorders but not for tuberous sclerosis (TSC) yet, which is an autosomal dominant disease caused by various variations in TSC1 or TSC2 gene. We aimed to explore the feasibility of NIPD on TSC. METHODS: We recruited singleton pregnancies at risk of TSC from 14 families with a proband child. Definitive NIPD for TSC was performed using targeted next-generation sequencing of cfDNA in parallel with maternal white blood cell DNA (wbcDNA). The NIPD results were validated by amniocentesis or postnatal gene testing and follow-up of the born children. RESULTS: Missense mutations, nonsense mutations, frameshift mutations, and splice-site variants which were obtained through de-novo, maternal, or paternal inheritance were included. The mean and minimum gestational weeks of NIPD were 17.18 ± 5.83 and 8 weeks, respectively. The NIPD results were 100% consistent with the amniocentesis or postnatal gene testing and follow-up of the born children. CONCLUSION: This study demonstrates that NIPD based on cfDNA is feasible for TSC, but required to be confirmed with more samples. Studies on TSC can contribute to the application and promotion of NIPD for monogenic disorders.

Rarity of fetal cells in exocervical samples for noninvasive prenatal diagnosis.

OBJECTIVES: The possibility to isolate fetal cells from pregnant women cervical samples has been discussed for five decades but is not currently applied in clinical practice. This study aimed at offering prenatal genetic diagnosis from fetal cells obtained through noninvasive exocervical sampling and immuno-sorted based on expression of HLA-G. METHODS: We first developed and validated robust protocols for cell detection and isolation on control cell lines expressing (JEG-3) or not (JAR) the HLA-G antigen, a specific marker for extravillous trophoblasts. We then applied these protocols to noninvasive exocervical samples collected from pregnant women between 6 and 14 weeks of gestational age. Sampling was performed through insertion and rotation of a brush at the ectocervix close to the external os of the endocervical canal. Finally, we attempted to detect and quantify trophoblasts in exocervical samples from pregnant women by ddPCR targeting the male SRY locus. RESULTS: For immunohistochemistry, a strong specific signal for HLA-G was observed in the positive control cell line and for rare cells in exocervical samples, but only in non-fixative conditions. HLA-G positive cells diluted in HLA-G negative cells were isolated by flow cytometry or magnetic cell sorting. However, no HLA-G positive cells could be recovered from exocervical samples. SRY gene was detected by ddPCR in exocervical samples from male (50%) but also female (27%) pregnancies. CONCLUSIONS: Our data suggest that trophoblasts are too rarely and inconstantly present in noninvasive exocervical samples to be reliably retrieved by standard immunoisolation techniques and therefore cannot replace the current practice for prenatal screening and diagnosis.

Significance of Low Maternal Serum Β-hCG Levels in the Assessment of the Risk of Atypical Chromosomal Abnormalities.

INTRODUCTION: The introduction of prenatal cell-free DNA as a screening test has surpassed traditional combined first-trimester screening (cFTS) in the detection of common trisomies. However, its current limitation in detecting only common trisomies is affecting the diagnostic yield for other clinically significant chromosomal abnormalities. METHODS: In efforts to optimize the detection of fetuses with genetic abnormalities, we have analyzed the relationship between the cFTS risk score and biomarkers with atypical chromosomal abnormalities. Furthermore, we have evaluated the impact of prenatal cell-free DNA screening on the detection of chromosomal abnormalities in our population. For these purposes, we performed a retrospective study of 877 singleton pregnancies who underwent chromosomal microarray analysis (CMA) between 2013 and 2020 and for whom cFTS data were available. RESULTS: The results demonstrated that low levels of free beta-human chorionic gonadotropin (ß-hCG) (≤0.37 multiples of the median) and increased fetal nuchal translucency (NT) (≥3.5 mm) were statistically associated with the presence of atypical chromosomal abnormalities. In fact, the risk of pathogenic CMA results increased from 6 to 10% when fetal NT was increased and from 6 to 20% when a low serum ß-hCG level was detected in the high-risk cFTS group. Moreover, our results showed that altered serum levels of ß-hCG can have a substantial impact on the early detection of clinically relevant copy number variants. DISCUSSION/CONCLUSION: Traditional cFTS can potentially identify a substantial proportion of atypical chromosomal aberrations, and women with increased NT or low maternal serum ß-hCG levels are at increased risk of having pathogenic CMA results. Our results may help clinicians and women decide whether invasive testing or prenatal cell-free DNA screening testing is more appropriate for each situation.

Haplotype-Based Noninvasive Prenatal Diagnosis of 21 Families With Duchenne Muscular Dystrophy: Real-World Clinical Data in China.

Noninvasive prenatal diagnosis (NIPD) of single-gene disorders has recently become the focus of clinical laboratories. However, reports on the clinical application of NIPD of Duchenne muscular dystrophy (DMD) are limited. This study aimed to evaluate the detection performance of haplotype-based NIPD of DMD in a real clinical environment. Twenty-one DMD families at 7-12 weeks of gestation were prospectively recruited. DNA libraries of cell-free DNA from the pregnant and genomic DNA from family members were captured using a custom assay for the enrichment of DMD gene exons and spanning single-nucleotide polymorphisms, followed by next-generation sequencing. Parental haplotype phasing was based on family linkage analysis, and fetal genotyping was inferred using the Bayes factor through target maternal plasma sequencing. Finally, the entire experimental process was promoted in the local clinical laboratory. We recruited 13 complete families, 6 families without paternal samples, and 2 families without probands in which daughter samples were collected. Two different maternal haplotypes were constructed based on family members in all 21 pedigrees at as early as 7 gestational weeks. Among the included families, the fetal genotypes of 20 families were identified at the first blood collection, and a second blood collection was performed for another family due to low fetal concentration. The NIPD result of each family was reported within 1 week. The fetal fraction in maternal cfDNA ranged from 1.87 to 11.68%. In addition, recombination events were assessed in two fetuses. All NIPD results were concordant with the findings of invasive prenatal diagnosis (chorionic villus sampling or amniocentesis). Exon capture and haplotype-based NIPD of DMD are regularly used for DMD genetic diagnosis, carrier screening, and noninvasive prenatal diagnosis in the clinic. Our method, haplotype-based early screening for DMD fetal genotyping via cfDNA sequencing, has high feasibility and accuracy, a short turnaround time, and is inexpensive in a real clinical environment.

Noninvasive prenatal diagnosis of duchenne muscular dystrophy in five Chinese families based on relative mutation dosage approach.

BACKGROUND: Relative haplotype dosage (RHDO) approach has been applied in noninvasive prenatal diagnosis (NIPD) of Duchenne muscular dystrophy (DMD). However, the RHDO procedure is relatively complicated and the parental haplotypes need to be constructed. Furthermore, it is not suitable for the diagnosis of de novo mutations or mosaicism in germ cells. Here, we investigated NIPD of DMD using a relative mutation dosage (RMD)-based approach-cell-free DNA Barcode-Enabled Single-Molecule Test (cfBEST), which has not previously been applied in the diagnosis of exon deletion. METHODS: Five DMD families caused by DMD gene point mutations or exon deletion were recruited for this study. After the breakpoints of exon deletion were precisely mapped with multiple PCR, the genotypes of the fetuses from the five DMD families were inferred using cfBEST, and were further validated by invasive prenatal diagnosis. RESULTS: The cfBEST results of the five families indicated that one fetus was female and did not carry the familial molecular alteration, three fetuses were carriers and one was male without the familial mutation. The invasive prenatal diagnosis results were consistent with those of the cfBEST procedure. CONCLUSION: This is the first report of NIPD of DMD using the RMD-based approach. We extended the application of cfBEST from point mutation to exon deletion mutation. The results showed that cfBEST would be suitable for NIPD of DMD caused by different kinds of mutation types.

Noninvasive prenatal diagnosis of monogenic disorders based on direct haplotype phasing through targeted linked-read sequencing.

BACKGROUND: Though massively parallel sequencing has been widely applied to noninvasive prenatal screen for common trisomy, the clinical use of massively parallel sequencing to noninvasive prenatal diagnose monogenic disorders is limited. This study was to develop a method for directly determining paternal haplotypes for noninvasive prenatal diagnosis of monogenic disorders without requiring proband's samples. METHODS: The study recruited 40 families at high risk for autosomal recessive diseases. The targeted linked-read sequencing was performed on high molecular weight (HMW) DNA of parents using customized probes designed to capture targeted genes and single-nucleotide polymorphisms (SNPs) distributed within 1Mb flanking region of targeted genes. Plasma DNA from pregnant mothers also underwent targeted sequencing using the same probes to determine fetal haplotypes according to parental haplotypes. The results were further confirmed by invasive prenatal diagnosis. RESULTS: Seventy-eight parental haplotypes of targeted gene were successfully determined by targeted linked-read sequencing. The predicted fetal inheritance of variant was correctly deduced in 38 families in which the variants had been confirmed by invasive prenatal diagnosis. Two families were determined to be no-call. CONCLUSIONS: Targeted linked-read sequencing method demonstrated to be an effective means to phase personal haplotype for noninvasive prenatal diagnosis of monogenic disorders.

The Impact of Cell-Free DNA Analysis on the Management of Retinoblastoma.

Retinoblastoma is a childhood eye cancer, mainly caused by mutations in the RB1 gene, which can be somatic or constitutional. Unlike many other cancers, tumour biopsies are not performed due to the risk of tumour dissemination. As a result, until recently, somatic genetic analysis was only possible if an affected eye was removed as part of a treatment. Several recent proof of principle studies have demonstrated that the analysis of tumour-derived cell-free DNA, either obtained from ocular fluid or blood plasma, has the potential to advance the diagnosis and influence the prognosis of retinoblastoma patients. It has been shown that a confirmed diagnosis is possible in retinoblastoma patients undergoing conservative treatment. In vivo genetic analysis of retinoblastoma tumours is also now possible, allowing the potential identification of secondary genetic events as prognostic biomarkers. In addition, noninvasive prenatal diagnosis in children at risk of inheriting retinoblastoma has been developed. Here, we review the current literature and discuss the potential impact of cell-free DNA analysis on both the diagnosis and treatment of retinoblastoma patients and their families.