Performance of the neoBona test: a new paired-end massively parallel shotgun sequencing approach for cell-free DNA-based aneuploidy screening.

OBJECTIVE: To assess the performance of screening for fetal trisomies 21, 18 and 13 by cell-free (cf) DNA analysis of maternal blood using a new method based on paired-end massively parallel shotgun sequencing (MPSS). METHODS: This was a blinded study of plasma samples (1mL) obtained from 1000 women undergoing screening for fetal trisomies 21, 18 and 13 at 11-13 weeks' gestation. The study included 50 cases with confirmed fetal trisomy 21, 30 with trisomy 18, 10 with trisomy 13 and 910 unaffected pregnancies. Paired-end MPSS with the neoBona® test allowed simultaneous assessment of fetal fraction, cfDNA fragment size distribution and chromosome counting, which were integrated into a new analysis algorithm to calculate trisomy likelihood ratios (t-score) for each chromosome of interest. Each sample was classified as trisomic or unaffected using chromosome-specific cut-offs set at t-score values of 1.5 for trisomy 21 and 3.0 for trisomies 18 and 13. RESULTS: Valid results were provided for 988 (98.8%) cases; 12 (1.2%) samples, from nine euploid and three trisomy 21 pregnancies, did not pass quality-control criteria and were excluded from further analysis. All 47 cases of trisomy 21, all 10 of trisomy 13, 29 of 30 with trisomy 18 and all 901 unaffected cases were classified correctly. Median fetal fraction was 10.5% (range, 0.3-33.8%) and trisomic and unaffected cases with low fetal fractions of < 1% were identified correctly. CONCLUSIONS: This novel method for cfDNA analysis of maternal plasma, which utilizes paired-end MPSS, can provide accurate prediction of fetal trisomies. Use of a new multicomponent t-score removes the need to reject samples with fetal fraction < 4%, which potentially extends the benefits of non-invasive prenatal cfDNA analysis to a larger proportion of pregnancies. © 2016 Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Performance of screening for aneuploidies by cell-free DNA analysis of maternal blood in twin pregnancies.

OBJECTIVES: To report clinical implementation of cell-free DNA (cfDNA) analysis of maternal blood in screening for trisomies 21, 18 and 13 in twin pregnancies and examine variables that could influence the failure rate of the test. METHODS: cfDNA testing was performed in 515 twin pregnancies at 10-28 weeks' gestation. The failure rate of the test to provide results was compared with that in 1847 singleton pregnancies, and logistic regression analysis was used to determine which factors among maternal and pregnancy characteristics were significant predictors of test failure. RESULTS: Failure rate of the cfDNA test at first sampling was 1.7% in singletons and 5.6% in twins. Of those with a test result, the median fetal fraction in twins was 8.7% (range, 4.1-30.0%), which was lower than that in singletons (11.7% (range, 4.0-38.9%)). Multivariable regression analysis demonstrated that twin pregnancy, higher maternal weight and conception by in-vitro fertilization provided significant independent prediction of test failure. Follow-up was available in 351 (68.2%) of the twin pregnancies and comprised 334 with euploid fetuses, 12 discordant for trisomy 21 and five discordant for trisomy 18. In all 323 euploid cases with a result, the risk score for each trisomy was < 1:10 000. In 11 of the 12 cases with trisomy 21 and in the five with trisomy 18, the cfDNA test gave a high-risk result, but in one case of trisomy 21, the score was < 1:10 000. CONCLUSION: In twin pregnancies screening by cfDNA testing is feasible, but the failure rate is higher and detection rate may be lower than in singletons.

Clinical application of midtrimester non-invasive fetal RHD genotyping and identification of RHD variants in a mixed-ethnic population.

OBJECTIVE: This study aims to assess the suitability of non-invasive prenatal RHD genotyping in non-immunized midtrimester pregnant women from a mixed ethnic population, to prevent unnecessary anti-D immunoglobulin prophylaxis and to identify RHD variants METHODS: Rhesus D-negative pregnant women were offered fetal RHD genotyping at 24 gestational weeks. A total of 284 samples were tested for RHD status using multiplex rt-PCR amplification of exons 5 and 7 of the RHD gene and exons 6 and 10 in selected cases. Women carrying RHD-negative fetuses were counseled about their option to avoid routine antenatal anti-D immunoglobulin administration. Diagnostic accuracy of RHD genotyping was compared with postnatal Rhesus D serotyping. RESULTS: A total of 184 positives (65%), 91 negatives (32%) and 7 cases (2.5%) compatibles with RHD variants were detected by RHD genotyping. No false negative results were found, and a single false positive was observed in a twin pregnancy. Genotyping was accepted when offered by 94% of women (284/302), and anti-D immunoglobulin was avoided in 95% (90/95) of RHD-negative fetuses. CONCLUSIONS: Non-invasive routine antenatal RHD genotyping at 24 weeks of pregnancy is a highly accurate method, resulting in the avoidance of 95% of unnecessary administrations of anti-D immunoglobulin, with no false negative results.

Supernumerary ring chromosome: an etiology for Pallister-Killian syndrome?

Characterization of marker chromosomes before the introduction of array CGH (aCGH) assays was only based on their banding patterns (G, C, and NOR staining) and fluorescent in situ hybridization techniques. The use of aCGH greatly improves the identification of marker chromosomes in some cases. We describe an atypical case of Pallister-Killian syndrome (PKS) detected at prenatal diagnosis with a very unusual cytogenetic presentation: a supernumerary ring chromosome including two copies of 12p. A similar anomaly described in a postnatal patient suggests ring chromosome as a possible cause of PKS. Extra ring chromosomes might be a more common etiology for PKS than previously thought, given the difficulty in their characterization before the advent of aCGH.

Protoneutron star cooling with convection: the effect of the symmetry energy.

We model neutrino emission from a newly born neutron star subsequent to a supernova explosion to study its sensitivity to the equation of state, neutrino opacities, and convective instabilities at high baryon density. We find the time period and spatial extent over which convection operates is sensitive to the behavior of the nuclear symmetry energy at and above nuclear density. When convection ends within the protoneutron star, there is a break in the predicted neutrino emission that may be clearly observable.

Hafnia alvei pyelonephritis in a renal transplant recipient: case report and review of an under-recognized nosocomial pathogen.

We describe the first case to our knowledge of Hafnia alvei pyelonephritis in a renal transplant recipient. Clinicians should consider this under-recognized pathogen when clinically evaluating immunosuppressed patients with a history of invasive procedures.

Prenatal detection of unbalanced chromosomal rearrangements by array CGH.

BACKGROUND: Karyotype analysis has been the standard method for prenatal cytogenetic diagnosis since the 1970s. Although highly reliable, the major limitation remains the requirement for cell culture, resulting in a delay of as much as 14 days to obtaining test results. Fluorescent in situ hybridisation (FISH) and quantitative fluorescent PCR (QF-PCR) rapidly detect common chromosomal abnormalities but do not provide a genome wide screen for unexpected imbalances. Array comparative genomic hybridisation (CGH) has the potential to combine the speed of DNA analysis with a large capacity to scan for genomic abnormalities. We have developed a genomic microarray of approximately 600 large insert clones designed to detect aneuploidy, known microdeletion syndromes, and large unbalanced chromosomal rearrangements. METHODS: This array was tested alongside an array with an approximate resolution of 1 Mb in a blind study of 30 cultured prenatal and postnatal samples with microscopically confirmed unbalanced rearrangements. RESULTS: At 1 Mb resolution, 22/30 rearrangements were identified, whereas 29/30 aberrations were detected using the custom designed array, owing to the inclusion of specifically chosen clones to give increased resolution at genomic loci clinically implicated in known microdeletion syndromes. Both arrays failed to identify a triploid karyotype. Thirty normal control samples produced no false positive results. CONCLUSIONS: Analysis of 30 uncultured prenatal samples showed that array CGH is capable of detecting aneuploidy in DNA isolated from as little as 1 ml of uncultured amniotic fluid; 29/30 samples were correctly diagnosed, the exception being another case of triploidy. These studies demonstrate the potential for array CGH to replace conventional cytogenetics in the great majority of prenatal diagnosis cases.

Double isochromosome 8q as single cytogenetic aberration in acute myelo-monocytic leukemia.

We report a case of double isochromosome 8q as a single cytogenetic abnormality in a patient with acute myelo-monocytic leukemia. Similarly to rare cases with tetrasomy 8, the patient showed monocytic involvement and was refractory to cytotoxic chemotherapy. We conclude that this kind of cytogenetic aberration is probably associated with distinct morphologic and clinical characteristics.

New molecular method for the detection of human papillomavirus type 16 integration.

Human papillomavirus (HPV) infection is the cause of cervical cancer. Integration of HPV-16 DNA in cervical cells is considered to be a key event in the progression towards invasive cancer, but little is known about this event in anal carcinogenesis. The integration could be a useful biomarker for cancer progression. Optimized assays are needed to determine the value of real-time detection of HPV integration in longitudinal studies, and this approach is only possible with a high-throughput assay. The aim of this study was to develop a new multiplex real-time PCR assay based on simultaneous amplification of the E2 and E6 HPV open reading frames (ORFs) in order to assess the physical status (episomal and/or integrated) of HPV-16 in anal cells of HIV-positive men. The comparative threshold (Ct) cycle values for E2 and E6 obtained for SiHA cells and artificial mixtures of episomal and integrated DNA were as expected: similar Ct for episomal forms and absence of E2 amplification for integrated forms. The multiplex real-time PCR was tested in 77 consecutive samples from individual HIV-infected patients with HPV-16 anal infection. The integration of HPV-16 was detected in 25 (32%) patients: 23 as mixed (episomal and integrated) and two as completed integrated forms. The integration occurs in the early stage of anal lesions and was associated with the severity of the lesions (p 0.004). The multiplex real-time PCR assay developed in the course of this study was shown to be a simple, sensitive, specific and inexpensive technique which may be applied routinely to detect HPV-16 integration.

Válvula nasal interna y rinoplastia estética / Internal nasal valve and aesthetic rhinoplasty

Se distingue en las fosas nasales la presencia de un estrecho o válvula y, de ella, se investiga específi camente su porción anterior conocida en la Especialidad de Cirugía Plástica como válvula nasal interna (VNI). Las características, disposición y relaciones de sus distintos componentes son analizados en esta presentación, puntualizando específi camente el singular comportamiento del mucoepitelio valvular con respecto a los cartílagos triangulares y cuadrangular. Se advierte sobre la posibilidad de complicaciones posoperatorias en el caso de indebido trato valvular. Se adjuntan preparados y sus correspondientes dibujos con el propósito de facilitar la comprensión de los hechos observados

Stands out the presence of a strait or valve in the nostrils, and about it, it's specifi cally investigated it's anterior portion known in the fi eld as internal nasal valve (INV). Characteristics, provision and relations of its individual components are discussed in this presentation, specifi cally pointing out the ubique behavior of the valvular mucosal epithelium in connection with the triangular and quadrangular cartilage. It warns out the possibility of post operative complications in case of improper valvular treatment. Attached preparations and their corresponding drawings in orden te facilitate the understanding of the observed facts.

How Magnetic is the Dirac Neutrino?

We derive model-independent, "naturalness" upper bounds on the magnetic moments munu of Dirac neutrinos generated by physics above the scale of electroweak symmetry breaking. In the absence of fine-tuning of effective operator coefficients, we find that current information on neutrino mass implies that[EQUATION: SEE TEXT] bohr magnetons. This bound is several orders of magnitude stronger than those obtained from analyses of solar and reactor neutrino data and astrophysical observations.

Isospin violation in epsilon'.

On the basis of a next-to-leading-order calculation in chiral perturbation theory, the first complete analysis of isospin breaking for direct CP violation in K0-->2 pi decays is performed. We find a destructive interference between three different sources of isospin violation in the CP violation parameter epsilon'. Within the uncertainties of large-N(c) estimates for the low-energy constants, the isospin violating correction for epsilon' is below 15%.

Neutrinoless double Beta decay and lepton flavor violation.

We point out that extensions of the standard model with low scale (approximately TeV) lepton number violation (LNV) generally lead to a pattern of lepton flavor violation (LFV) experimentally distinguishable from the one implied by models with grand unified theory scale LNV. As a consequence, muon LFV processes provide a powerful diagnostic tool to determine whether or not the effective neutrino mass can be deduced from the rate of neutrinoless double beta decay. We discuss the role of mu-->egamma and mu-->e conversion in nuclei, which will be studied with high sensitivity in forthcoming experiments.

Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells.

We have refined polymerase chain reaction (PCR) assays for the detection of sickle cell anaemia, the delta F 508 deletion causing cystic fibrosis, and the IVS1-110 mutation leading to beta thalassaemia, allowing them to be successfully performed upon single cells using fluorescent primers. We have also assessed the possibility of detecting aneuploidies of chromosomes 13, 18 and 21 using a quantitative fluorescent polymerase chain reaction (QF-PCR) with primers flanking polymorphic short tandem repeat (STR) markers. Trisomies were readily diagnosed by the detection of tri-allelic patterns. However some heterozygote normal and trisomic diallelic patterns did not produce the expected ratios of amplified PCR products due to preferential DNA sequence amplification. Total allelic drop out (ADO) did not occur with any of the cells tested. Multiplex QF-PCR assays can be performed on a single cell in under 6 h and simultaneously provide diagnosis of single gene defects, sex determination and an indication of selected chromosome aneuploidy.

Assessment of new markers for the rapid detection of aneuploidies by quantitative fluorescent PCR (QF-PCR).

Rapid prenatal diagnoses of major chromosome aneuploidies have been achieved successfully using quantitative fluoresent PCR (QF-PCR) assays and small tandem repeat (STR) markers. Here we report the results of evaluating the use of previously untested X-linked STRs, (DXS6803) and (DXS6809), together with modified amelogenin (AMXY) sequences and the X22 marker that maps in the pseudoautosomal region PAR2 on the long arm of the X and Y chromosomes. These markers will allow prenatal diagnoses of sex chromosome aneuploidies such as 45,X (pure Turner Syndrome), 47,XXY and 47,XYY, while assessing the sex of the fetuses. Data are also presented concerning the difficulties associated with the evaluation of the frequencies of the various types of sub-populations of cells in amniotic fluid samples collected from fetuses with sex chromosome mosaicism. The results of evaluating the use of new markers for the rapid diagnosis of aneuploidies affecting chromosomes 21,18 and 13 are also presented. Three chromosome 21 specific STRs have been found to produce trisomic triallelic or diallelic patterns from all amniotic samples retrieved from fetuses with Down Syndrome. Since all samples tested were amplified and no false positive or negative results were observed, the present results confirm the diagnostic value of QF-PCR for the prenatal detection of major numerical chromosome disorders.

Rapid detection of chromosomes X and Y aneuploidies by quantitative fluorescent PCR.

Quantitative fluorescent polymerase chain reaction (QF-PCR) assays and small tandem repeat (STR) markers have been successfully employed for the rapid detection of major numerical aneuploidies affecting human autosomes. So far, the analysis of chromosomes X and Y disorders has been hampered by the rarity of highly polymorphic markers which could distinguish normal female homozygous PCR patterns from those seen in patients with Turner's syndrome. A new marker (X22) of the X/Y chromosomes has been identified which maps in the Xq/Yq pseudoautosomal region PAR2; used together with the HPRT it allows the rapid diagnosis of numerical aneuploidies of the sex chromosomes. Blood samples from normal male and female subjects and from patients with X and Y chromosome disorders (45,X and 47, XXY) have been tested by QF-PCR with the X22 polymorphic pentanucleotide (12 alleles) together with the HPRT and P39 markers. The samples were also tested by multiplex QF-PCR with STRs specific for chromosomes 21,18,13 and amelogenin (AMXY). Tested by QF-PCR, all samples from normal females were heterozygous for either the X22 or the HPRT marker with fluorescent peak ratios near 1:1, thus allowing a correct, rapid diagnosis of their chromosome complement. Turner's patients (45,X) showed only one X22 and one HPRT fluorescent peak, thus documenting the presence of a single X chromosome. Turner's patients with mosaicism showed a major fluorescent peak for the X22 and HPRT markers and a minor peak revealing the presence of a second minor population of cells. Two 47, XXY cases could also be diagnosed. Multiplex analyses can be performed using simultaneously STR markers for chromosomes 21,18,13 X and Y. The diagnostic value of a third X-linked marker (P39) was also investigated. These results suggest that rapid diagnosis of major numerical anomalies of the X and Y chromosomes can be performed using QF-PCR with a new highly polymorphic X-linked marker, X22, which maps in the Xq/Yq pseudoautosomal region PAR 2. Multiplex QF-PCR tests-using the X22 STR in association with HPRT and, in rare cases, a third P39 marker-allow the rapid diagnosis of major aneuploidies affecting chromosomes 21, 18, 13, X and Y. The X22 marker can also be employed for the detection of fetal cells present in maternal peripheral blood or the endocervical canal.