RESUMO
The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.
Assuntos
Células-Tronco Adultas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Células Germinativas/citologia , Fator 4 Semelhante a Kruppel , Antígenos CD15/metabolismo , Camundongos , Miócitos Cardíacos/citologiaRESUMO
BACKGROUND: Noninvasive prenatal testing (NIPT) uses cell-free DNA (cfDNA) as an analyte to detect copy-number alterations in the fetal genome. Because maternal and fetal cfDNA contributions are comingled, changes in the maternal genome can manifest as abnormal NIPT results. Circulating tumor DNA (ctDNA) present in cases of maternal neoplasia has the potential to distort the NIPT readout to a degree that prevents interpretation, resulting in a nonreportable test result for fetal aneuploidy. METHODS: NIPT cases that showed a distortion from normal euploid genomic representation were communicated to the caregiving physician as nonreportable for fetal aneuploidy. Follow-up information was subsequently collected for these cases. More than 450000 pregnant patients who submitted samples for clinical laboratory testing >3 years are summarized. Additionally, in-depth analysis was performed for >79000 research-consented samples. RESULTS: In total, 55 nonreportable NIPT cases with altered genomic profiles were cataloged. Of these, 43 had additional information available to enable follow-up. A maternal neoplasm was confirmed in 40 of these cases: 18 malignant, 20 benign uterine fibroids, and 2 with radiological confirmation but without pathological classification. CONCLUSIONS: In a population of pregnant women who submitted a blood sample for cfDNA testing, an abnormal genomic profile not consistent with fetal abnormalities was detected in about 10 out of 100000 cases. A subset of these observations (18 of 43; 41.9%) was attributed to maternal malignant neoplasms. These observational results suggest the need for a controlled trial to evaluate the potential of using cfDNA as an early biomarker of cancer.
Assuntos
Ácidos Nucleicos Livres/sangue , Achados Incidentais , Complicações Neoplásicas na Gravidez/diagnóstico , Diagnóstico Pré-Natal/métodos , Adulto , DNA Tumoral Circulante/sangue , Estudos de Coortes , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Gravidez , Complicações Neoplásicas na Gravidez/sangueRESUMO
BACKGROUND: Current methods for noninvasive prenatal testing (NIPT) ascertain fetal aneuploidies using either direct counting measures of DNA fragments from specific genomic regions or relative measures of single nucleotide polymorphism frequencies. Alternatively, the ratios of paralogous sequence pairs were predicted to reflect fetal aneuploidy. We developed a NIPT assay that uses paralog sequences to enable noninvasive detection of fetal trisomy 21 (T21) and trisomy 18 (T18) using cell-free DNA (cfDNA) from maternal plasma. METHODS: A total of 1060 primer pairs were designed to determine fetal aneuploidy status, fetal sex, and fetal fraction. Each library was prepared from cfDNA by coamplifying all 1060 target pairs together in a single reaction well. Products were measured using massively parallel sequencing and deviations from expected paralog ratios were determined based on the read depth from each paralog. RESULTS: We evaluated this assay in a blinded set of 480 cfDNA samples with fetal aneuploidy status determined by the MaterniT21® PLUS assay. Samples were sequenced (mean = 2.3 million reads) with 432 samples returning a result. Using the MaterniT21 PLUS assay for paired plasma aliquots from the same individuals as a reference, all 385 euploid samples, all 31 T21 samples, and 14 of 16 T18 samples were detected with no false positive results observed. CONCLUSIONS: This study introduces a novel NIPT aneuploidy detection approach using targeted sequencing of paralog motifs and establishes proof-of-concept for a potentially low-cost, highly scalable method for the identification of selected fetal aneuploidies with performance and nonreportable rate similar to other published methods.
Assuntos
Aneuploidia , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Diagnóstico Pré-Natal , Análise de Sequência de DNA , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 21/genética , DNA/análise , HumanosRESUMO
BACKGROUND: The development of sequencing-based noninvasive prenatal testing (NIPT) has been largely focused on whole-chromosome aneuploidies (chromosomes 13, 18, 21, X, and Y). Collectively, they account for only 30% of all live births with a chromosome abnormality. Various structural chromosome changes, such as microdeletion/microduplication (MD) syndromes are more common but more challenging to detect. Recently, several publications have shown results on noninvasive detection of MDs by deep sequencing. These approaches demonstrated the proof of concept but are not economically feasible for large-scale clinical applications. METHODS: We present a novel approach that uses low-coverage whole genome sequencing (approximately 0.2×) to detect MDs genome wide without requiring prior knowledge of the event's location. We developed a normalization method to reduce sequencing noise. We then applied a statistical method to search for consistently increased or decreased regions. A decision tree was used to differentiate whole-chromosome events from MDs. RESULTS: We demonstrated via a simulation study that the sensitivity difference between our method and the theoretical limit was <5% for MDs ≥9 Mb. We tested the performance in a blinded study in which the MDs ranged from 3 to 40 Mb. In this study, our algorithm correctly identified 17 of 18 cases with MDs and 156 of 157 unaffected cases. CONCLUSIONS: The limit of detection for any given MD syndrome is constrained by 4 factors: fetal fraction, MD size, coverage, and biological and technical variability of the event region. Our algorithm takes these factors into account and achieved 94.4% sensitivity and 99.4% specificity.
Assuntos
Transtornos Cromossômicos/genética , DNA/genética , Diagnóstico Pré-Natal/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Transtornos Cromossômicos/sangue , Síndrome de Cri-du-Chat/sangue , DNA/sangue , Síndrome de DiGeorge/sangue , Feminino , Feto , Humanos , Limite de Detecção , Síndrome de Prader-Willi/sangue , Gravidez , Diagnóstico Pré-Natal/normas , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: This study introduces a novel method, referred to as SeqFF, for estimating the fetal DNA fraction in the plasma of pregnant women and to infer the underlying mechanism that allows for such statistical modeling. METHODS: Autosomal regional read counts from whole-genome massively parallel single-end sequencing of circulating cell-free DNA (ccfDNA) from the plasma of 25 312 pregnant women were used to train a multivariate model. The pretrained model was then applied to 505 pregnant samples to assess the performance of SeqFF against known methodologies for fetal DNA fraction calculations. RESULTS: Pearson's correlation between chromosome Y and SeqFF for pregnancies with male fetuses from two independent cohorts ranged from 0.932 to 0.938. Comparison between a single-nucleotide polymorphism-based approach and SeqFF yielded a Pearson's correlation of 0.921. Paired-end sequencing suggests that shorter ccfDNA, that is, less than 150 bp in length, is nonuniformly distributed across the genome. Regions exhibiting an increased proportion of short ccfDNA, which are more likely of fetal origin, tend to provide more information in the SeqFF calculations. CONCLUSION: SeqFF is a robust and direct method to determine fetal DNA fraction. Furthermore, the method is applicable to both male and female pregnancies and can greatly improve the accuracy of noninvasive prenatal testing for fetal copy number variation.
Assuntos
DNA/sangue , Feto , Sequenciamento de Nucleotídeos em Larga Escala , Testes para Triagem do Soro Materno/métodos , Análise de Sequência de DNA/métodos , Sistema Livre de Células , Feminino , Humanos , Masculino , Modelos Estatísticos , Análise Multivariada , Polimorfismo de Nucleotídeo Único , Gravidez , Estudos RetrospectivosRESUMO
We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.
Assuntos
Evolução Molecular Direcionada , Escherichia coli/genética , Genoma Bacteriano , Adaptação Fisiológica , Meios de Cultura , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/fisiologia , Genótipo , Glicerol/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Seleção Genética , Fatores de TempoRESUMO
PURPOSE: We sought to compare measurements of circulating cell-free DNA as well as Down syndrome test results in women with naturally conceived pregnancies with those conceived using assisted reproductive technologies. METHODS: Data regarding assisted reproductive technologies were readily available from seven enrollment sites participating in an external clinical validation trial of nested case/control design. Measurements of circulating cell-free fetal and total DNA, fetal fraction (ratio of fetal to total DNA), chromosome-specific z-scores, and karyotype results were available for analysis. RESULTS: Analyses were restricted to 632 euploid (5.2% assisted reproductive technologies) and 73 Down syndrome (13.7% assisted reproductive technologies), including 16 twin pregnancies. No differences were found for fetal or total circulating cell-free DNA, or for the fetal fraction in euploid (P = 0.70) or Down syndrome (P = 0.58) pregnancies by method of conception. There appeared to be systematic z-score reductions for chromosomes 21, 18, and 13 in assisted reproductive technologies versus natural euploid pregnancies (P = 0.048, 0.0032, and 0.36, respectively). CONCLUSION: Assisted reproductive technologies and naturally conceived pregnancies contribute similar levels of circulating cell-free DNA into maternal circulation. Small differences in the z-scores of pregnancies achieved by assisted reproductive technologies were observed and do not appear to be test-related artifacts. However, the findings need confirmation before any consideration of changes to testing and reporting protocols.
Assuntos
Aneuploidia , DNA/sangue , Síndrome de Down/epidemiologia , Síndrome de Down/genética , Técnicas de Reprodução Assistida/efeitos adversos , DNA/genética , Síndrome de Down/diagnóstico , Feminino , Testes Genéticos , Humanos , GravidezRESUMO
BACKGROUND: Massively parallel sequencing of circulating cell free (ccf) DNA from maternal plasma has been demonstrated to be a powerful method for the detection of fetal copy number variations (CNVs). Although the detection of CNVs has been described by multiple independent groups, genomic aberrations resulting in copy number-neutral events including balanced translocations have proven to be more challenging to detect noninvasively from ccf DNA. METHODS: Data modeling was initially performed to evaluate multiple methods, ultimately leveraging the short length of ccf DNA and paired-end sequencing to construct read-specific mapping characteristics. After testing in a model system, we evaluated the methods on ccf DNA isolated from the plasma of a donor known to be carrying a fetus with a balanced translocation [t(8;11)]. Sequencing was performed with Illumina sequencing technology. RESULTS: Our methodology identified the known translocation (P = 1.21 × 10(-8)) and discounted the likelihood of others, enabling the base specific identification of the rearrangement positions. In total, 402 unique sequencing reads spanned the putative breakpoints, of which 76 contained the structural rearrangement. In addition, 38 of the chimeric reads were mapped to each of the resulting derivative chromosomes, supporting the presence of a reciprocal translocation. Finally, we identified a 6-bp deletion present within der(8) that was absent from the der(11) reciprocal rearrangement. CONCLUSIONS: We have developed an algorithm to detect balanced rearrangements and applied our methodology to demonstrate the first proof-of-principle study on the noninvasive detection of a fetal-specific balanced translocation by sequencing ccf DNA from maternal plasma.
Assuntos
Variações do Número de Cópias de DNA/genética , DNA/sangue , Feto/metabolismo , Diagnóstico Pré-Natal/métodos , Translocação Genética , Adulto , Algoritmos , Sequência de Bases , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 8/genética , Simulação por Computador , DNA/genética , Feminino , Idade Gestacional , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Gravidez , Análise de Sequência de DNA/métodosRESUMO
OBJECTIVE: The objective of this study was to validate the clinical performance of massively parallel genomic sequencing of cell-free deoxyribonucleic acid contained in specimens from pregnant women at high risk for fetal aneuploidy to test fetuses for trisomies 21, 18, and 13; fetal sex; and the common sex chromosome aneuploidies (45, X; 47, XXX; 47, XXY; 47, XYY). STUDY DESIGN: This was a prospective multicenter observational study of pregnant women at high risk for fetal aneuploidy who had made the decision to pursue invasive testing for prenatal diagnosis. Massively parallel single-read multiplexed sequencing of cell-free deoxyribonucleic acid was performed in maternal blood for aneuploidy detection. Data analysis was completed using sequence reads unique to the chromosomes of interest. RESULTS: A total of 3430 patients were analyzed for demographic characteristics and medical history. There were 137 fetuses with trisomy 21, 39 with trisomy 18, and 16 with trisomy 13 for a prevalence rate of the common autosomal trisomies of 5.8%. There were no false-negative results for trisomy 21, 3 for trisomy 18, and 2 for trisomy 13; all 3 false-positive results were for trisomy 21. The positive predictive values for trisomies 18 and 13 were 100% and 97.9% for trisomy 21. A total of 8.6% of the pregnancies were 21 weeks or beyond; there were no aneuploid fetuses in this group. All 15 of the common sex chromosome aneuploidies in this population were identified, although there were 11 false-positive results for 45,X. Taken together, the positive predictive value for the sex chromosome aneuploidies was 48.4% and the negative predictive value was 100%. CONCLUSION: Our prospective study demonstrates that noninvasive prenatal analysis of cell-free deoxyribonucleic acid from maternal plasma is an accurate advanced screening test with extremely high sensitivity and specificity for trisomy 21 (>99%) but with less sensitivity for trisomies 18 and 13. Despite high sensitivity, there was modest positive predictive value for the small number of common sex chromosome aneuploidies because of their very low prevalence rate.
Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Testes para Triagem do Soro Materno , Análise de Sequência de DNA/métodos , Aberrações dos Cromossomos Sexuais , Transtornos dos Cromossomos Sexuais/diagnóstico , Trissomia/diagnóstico , Adulto , Transtornos Cromossômicos/diagnóstico , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 18 , Cromossomos Humanos X , Cromossomos Humanos Y , Síndrome de Down/diagnóstico , Feminino , Humanos , Valor Preditivo dos Testes , Gravidez , Estudos Prospectivos , Síndrome da Trissomia do Cromossomo 13 , Síndrome da Trissomía do Cromossomo 18RESUMO
Noninvasive prenatal testing (NIPT) uses cell-free fetal DNA from the plasma of pregnant women to provide valuable information about the potential risks for fetal aneuploidy. This article provides a historical overview of both invasive diagnostic testing and serum screening approaches, both biochemical and the newer molecular noninvasive prenatal testing assays, used to identify patients who would be best served by invasive testing.
Assuntos
Amniocentese/métodos , Amostra da Vilosidade Coriônica/métodos , DNA/análise , Síndrome de Down/diagnóstico , Testes Genéticos/métodos , Diagnóstico Pré-Natal/métodos , Amniocentese/história , Aneuploidia , Amostra da Vilosidade Coriônica/história , Síndrome de Down/genética , Feminino , História do Século XX , História do Século XXI , Humanos , GravidezRESUMO
OBJECTIVE: Whole-genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes. METHOD: Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype. A classification algorithm for SCA detection was constructed and trained on this cohort. Another study of 411 maternal samples from women with blinded-to-laboratory fetal karyotypes was then performed to determine the accuracy of the classification algorithm. RESULTS: In the training cohort, the new algorithm had a detection rate (DR) of 100% (95%CI: 82.3%, 100%), a false positive rate (FPR) of 0.1% (95%CI: 0%, 0.3%), and nonreportable rate of 6% (95%CI: 4.9%, 7.4%) for SCA determination. The blinded validation yielded similar results: DR of 96.2% (95%CI: 78.4%, 99.8%), FPR of 0.3% (95%CI: 0%, 1.8%), and nonreportable rate of 5% (95%CI: 3.2%, 7.7%) for SCA determination CONCLUSION: Noninvasive prenatal identification of the most common sex chromosome aneuploidies is possible using ccf DNA and massively parallel sequencing with a high DR and a low FPR.
Assuntos
Aneuploidia , Diagnóstico Pré-Natal/métodos , Análise de Sequência de DNA/métodos , Aberrações dos Cromossomos Sexuais , Cromossomos Humanos X/genética , Cromossomos Humanos Y/genética , Estudos de Coortes , DNA/sangue , DNA/genética , Feminino , Feto/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mães , Gravidez/sangueRESUMO
PURPOSE: To determine whether maternal plasma cell-free DNA sequencing can effectively identify trisomy 18 and 13. METHODS: Sixty-two pregnancies with trisomy 18 and 12 with trisomy 13 were selected from a cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported), and their samples tested using a laboratory-developed, next-generation sequencing test. Interpretation of the results for chromosome 18 and 13 included adjustment for CG content bias. RESULTS: Among the 99.1% of samples interpreted (1,971/1,988), observed trisomy 18 and 13 detection rates were 100% (59/59) and 91.7% (11/12) at false-positive rates of 0.28% and 0.97%, respectively. Among the 17 samples without an interpretation, three were trisomy 18. If z-score cutoffs for trisomy 18 and 13 were raised slightly, the overall false-positive rates for the three aneuploidies could be as low as 0.1% (2/1,688) at an overall detection rate of 98.9% (280/283) for common aneuploidies. An independent academic laboratory confirmed performance in a subset. CONCLUSION: Among high-risk pregnancies, sequencing circulating cell-free DNA detects nearly all cases of Down syndrome, trisomy 18, and trisomy 13, at a low false-positive rate. This can potentially reduce invasive diagnostic procedures and related fetal losses by 95%. Evidence supports clinical testing for these aneuploidies.
Assuntos
Cromossomos Humanos Par 13 , Cromossomos Humanos Par 18 , DNA/sangue , Síndrome de Down/diagnóstico , Análise de Sequência de DNA , Trissomia/diagnóstico , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Gravidez , Diagnóstico Pré-Natal , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estados Unidos , Adulto JovemRESUMO
BACKGROUND: Efforts have been undertaken recently to assess the fetal genome through analysis of circulating cell-free (ccf) fetal DNA obtained from maternal plasma. Sequencing analysis of such ccf DNA has been shown to enable accurate prenatal detection of fetal aneuploidies, including trisomies of chromosomes 21, 18, and 13. We sought to extend these analyses to examine subchromosomal copy number variants through the sequencing of ccf DNA. We examined a clinically relevant genomic region, chromosome 22q11.2, the location of a series of well-characterized deletion anomalies that cause 22q11.2 deletion syndrome. METHODS: We sequenced ccf DNA isolated from maternal plasma samples obtained from 2 patients with confirmed 22q11.2 deletion syndrome and from 14 women at low risk for fetal chromosomal abnormalities. The latter samples were used as controls, and the mean genomic coverage was 3.83-fold. Data were aligned to the human genome, repetitive regions were removed, the remaining data were normalized for GC content, and z scores were calculated for the affected region. RESULTS: The median fetal DNA contribution for all samples was 18%, with the affected samples containing 17%-18% fetal DNA. Using a technique similar to that used for sequencing-based fetal aneuploidy detection from maternal plasma, we detected a statistically significant loss of representation of a portion of chromosome 22q11.2 in both of the affected fetal samples. No such loss was detected in any of the control samples. CONCLUSIONS: Noninvasive prenatal diagnosis of subchromosomal fetal genomic anomalies is feasible with next-generation sequencing.
Assuntos
DNA/genética , Feto , Deleção Cromossômica , Cromossomos Humanos Par 22/genética , DNA/sangue , Estudos de Viabilidade , Feminino , Idade Gestacional , Humanos , Gravidez , Análise de Sequência de DNARESUMO
Acute myeloid leukemia (AML) is characterized by molecular heterogeneity that is not fully reflected in the current classification system. Recent insights point toward a significant role of aberrant DNA methylation in leukemogenesis. Therefore, we investigated the prognostic impact of DNA methylation in AML. To screen for promoter methylation in AML we applied a combination of base-specific cleavage biochemistry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a powerful methodology allowing for quantitatively investigating DNA methylation status in a large series of both promoter regions and leukemia samples. We analyzed 92 genomic regions in 182 patient samples, correlated findings with clinical and molecular data, and validated the results in an independent cohort of 74 AML samples. Using this approach, we were able to identify novel leukemia subgroups based on distinct DNA methylation patterns. Furthermore, we defined a methylation-based outcome predictor for patient survival (P < .01) that in multivariable analysis provided independent prognostic information (hazard ratio, 1.52; 95% CI, 1.06-2.16). Here, we report the first large-scale methylation-based outcome predictor in AML, and thereby our findings support the use of genomic methylation markers for improved molecular classification and prognostication in adult AML.
Assuntos
Metilação de DNA , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Análise de Sequência de DNA/métodos , Adulto , Cromossomos Humanos Par 17 , Ilhas de CpG/genética , Análise Citogenética/métodos , Metilação de DNA/fisiologia , DNA de Neoplasias/análise , Perfilação da Expressão Gênica/métodos , Humanos , Leucemia Mieloide Aguda/genética , Análise Multivariada , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Prognóstico , Regiões Promotoras Genéticas/genética , Análise de SobrevidaRESUMO
OBJECTIVE: Studies on prenatal testing for Down syndrome (trisomy 21), trisomy 18, and trisomy 13 by massively parallel shotgun sequencing (MPSS) of circulating cell free DNA have been, for the most part, limited to singleton pregnancies. If MPSS testing is offered clinically, it is important to know if these trisomies will also be identified in multiple pregnancies. METHOD: Among a cohort of 4664 high-risk pregnancies, maternal plasma samples were tested from 25 twin pregnancies (17 euploid, five discordant and two concordant for Down syndrome; one discordant for trisomy 13) and two euploid triplet pregnancies [Correction made here after initial online publication.]. Results were corrected for GC content bias. For each target chromosome (21, 18, and 13), z-scores of 3 or higher were considered consistent with trisomy. RESULTS: Seven twin pregnancies with Down syndrome, one with trisomy 13, and all 17 twin euploid pregnancies were correctly classified [detection rate 100%, 95% confidence interval (CI) 59%-100%, false positive rate 0%, 95% CI 0%-19.5%], as were the two triplet euploid pregnancies. CONCLUSION: Although study size is limited, the underlying biology combined with the present data provide evidence that MPSS testing can be reliably used as a secondary screening test for Down syndrome in women with high-risk twin gestations.
Assuntos
Síndrome de Down/sangue , Síndrome de Down/diagnóstico , Gravidez de Gêmeos/sangue , Trissomia/diagnóstico , Feminino , Humanos , Masculino , Gravidez , Gravidez de Trigêmeos/sangue , Análise de Sequência de DNARESUMO
PURPOSE: Prenatal screening for Down syndrome has improved, but the number of resulting invasive diagnostic procedures remains problematic. Measurement of circulating cell-free DNA in maternal plasma might offer improvement. METHODS: A blinded, nested case-control study was designed within a cohort of 4664 pregnancies at high risk for Down syndrome. Fetal karyotyping was compared with an internally validated, laboratory-developed test based on next-generation sequencing in 212 Down syndrome and 1484 matched euploid pregnancies. None had been previously tested. Primary testing occurred at a CLIA-certified commercial laboratory, with cross validation by a CLIA-certified university laboratory. RESULTS: Down syndrome detection rate was 98.6% (209/212), the false-positive rate was 0.20% (3/1471), and the testing failed in 13 pregnancies (0.8%); all were euploid. Before unblinding, the primary testing laboratory also reported multiple alternative interpretations. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving performance. CONCLUSION: When applied to high-risk pregnancies, measuring maternal plasma DNA detects nearly all cases of Down syndrome at a very low false-positive rate. This method can substantially reduce the need for invasive diagnostic procedures and attendant procedure-related fetal losses. Although implementation issues need to be addressed, the evidence supports introducing this testing on a clinical basis.
Assuntos
Síndrome de Down/diagnóstico , Doenças Fetais/diagnóstico , Diagnóstico Pré-Natal/métodos , Análise de Sequência de DNA/métodos , Adulto , Estudos de Casos e Controles , Método Duplo-Cego , Síndrome de Down/sangue , Síndrome de Down/genética , Reações Falso-Positivas , Feminino , Doenças Fetais/sangue , Doenças Fetais/genética , Humanos , Cariotipagem , Gravidez , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: The objective of the study was the evaluation of a novel multiplex assay to detect fetal Rh blood group D-antigen gene (RHD) loci in maternal plasma from RhD-negative, pregnant women. STUDY DESIGN: An RHD genotyping assay was designed to detect exons 4, 5, 7, and 10 and RHDΨ (pseudogene) of the RHD gene along with a Y chromosome-specific assay and a generic polymerase chain reaction amplification control. Plasma samples from 150 RhD-negative pregnant women were assayed for fetal RHD genotype using the MassARRAY system. RESULTS: The fetal RHD status of 148 of 150 samples (98.7%) was correctly classified; 86 (57.3%) and 62 (41.3%) were positive and negative, respectively. CONCLUSION: This study demonstrates that noninvasive prenatal diagnostics with a single-reaction multiplexed assay is a viable path toward routine characterization of fetal RHD genotypes using circulating cell-free fetal DNA in maternal plasma on the MassARRAY system and is perhaps preferable to serologic testing as currently used clinically.