Whole-genome sequencing in prenatally detected congenital malformations: prospective cohort study in clinical setting.

OBJECTIVE: To investigate the diagnostic yield of trio whole-genome sequencing (WGS) in fetuses with various congenital malformations referred to a tertiary center for prenatal diagnosis. METHODS: In this prospective study, 50 pregnancies with different congenital malformations, negative for trisomies and causative copy-number variants, were analyzed further with fetal-parental trio WGS analysis. Parents were eligible for inclusion if they accepted further investigation following the detection of isolated or multiple malformations on prenatal ultrasound. Cases with isolated increased nuchal translucency, gamete donation or multiple pregnancy were excluded. WGS with the Illumina Inc. 30× polymerase-chain-reaction-free short-read sequencing included analysis of single-nucleotide variants, insertions and deletions, structural variants, short tandem repeats and copy-number identification of SMN1 and SMN2 genes. RESULTS: A molecular diagnosis was achieved in 13/50 (26%) cases. Causative sequence variants were identified in 12 genes: FGFR3 (n = 2), ACTA1 (n = 1), CDH2 (n = 1), COL1A2 (n = 1), DHCR7 (n = 1), EYA1 (n = 1), FBXO11 (n = 1), FRAS1 (n = 1), L1CAM (n = 1), OFD1 (n = 1), PDHA1 (n = 1) and SOX9 (n = 1). The phenotypes of the cases were divided into different groups, with the following diagnostic yields: skeletal malformation (4/9 (44%)), multisystem malformation (3/7 (43%)), central nervous system malformation (5/15 (33%)) and thoracic malformation (1/10 (10%)). Additionally, two cases carried variants that were considered potentially clinically relevant, even though they were assessed as variants of uncertain significance, according to the guidelines provided by the American College of Medical Genetics and Genomics. Overall, we identified a causative or potentially clinically relevant variant in 15/50 (30%) cases. CONCLUSIONS: We demonstrate a diagnostic yield of 26% with clinical WGS in prenatally detected congenital malformations. This study emphasizes the benefits that WGS can bring to the diagnosis of fetal structural anomalies. It is important to note that causative chromosomal aberrations were excluded from our cohort before WGS. As chromosomal aberrations are a well-known cause of prenatally detected congenital malformations, future studies using WGS as a primary diagnostic test, including assessment of chromosomal aberrations, may show that the detection rate exceeds the diagnostic yield of this study. WGS can add clinically relevant information, explaining the underlying cause of the fetal anomaly, which will provide information concerning the specific prognosis of the condition, as well as estimate the risk of recurrence. A genetic diagnosis can also provide more reproductive choice for future pregnancies. © 2024 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Diagnostic yield using whole-genome sequencing and in-silico panel of 281 genes associated with non-immune hydrops fetalis in clinical setting.

OBJECTIVE: To investigate the diagnostic yield of clinical whole-genome sequencing (WGS) in prenatally diagnosed non-immune hydrops fetalis (NIHF). METHODS: This was a retrospective study of 23 fetuses with prenatally diagnosed NIHF, negative for trisomies and copy-number variants, referred for analysis by WGS with an in-silico panel of 281 genes associated with hydrops fetalis. Due to identification of a high proportion of causative variants in the HRAS gene in the main cohort, Sanger sequencing of HRAS was performed in a replication cohort, consisting of 24 additional fetuses with NIHF that were negative for trisomies and copy-number variants and had not undergone WGS. RESULTS: Of the 23 fetuses in the main cohort, a molecular diagnosis was achieved in 12 (52.2%). Pathogenic or likely pathogenic variants were identified in seven genes: HRAS (n = 5), RIT1 (n = 2), FOXP3 (n = 1), GLB1 (n = 1), MAP2K1 (n = 1), PTPN11 (n = 1) and RASA1 (n = 1). The inheritance pattern of the 12 causative variants was autosomal dominant in 10 cases (HRAS, MAP2K1, PTPN11, RASA1, RIT1), autosomal recessive in one (GLB1) and X-linked recessive in one (FOXP3). Of the 24 fetuses in the replication cohort, a pathogenic variant in HRAS was identified in one, resulting in an overall frequency of causative HRAS variants of 12.8% (6/47) in our two cohorts. CONCLUSIONS: We demonstrate a diagnostic yield of 52% with clinical WGS in NIHF using an in-silico panel of 281 genes. However, the high diagnostic yield may be attributed to the small sample size and possible over-representation of severe phenotypes in the included fetuses. Bearing in mind that chromosomal abnormalities were excluded in our cohorts, a detection rate of up to 75% is possible in prenatally diagnosed NIHF when WGS analysis includes calling of chromosomal aberrations. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Experimental and computational study of species formed during electrochemical stripping oxidation of copper in chloride media determination of copper(II) in the ng l(-1) range by stripping potentiometry.

A novel glassy carbon electrode design, permitting medium exchange in batch mode without loss of electrode potential control, has been used for the study of copper(I) and copper(II) species formed during constant current stripping oxidation of copper in chloride media. It was found that copper(II) species dominated at chloride concentrations below about 1 mM and that soluble copper(I) species dominated at chloride concentrations above about 100 mM. In the concentration range 1-100 mM, soluble copper(I) and copper(II) species are formed as well as solid copper(I) chloride, the latter giving rise to a split peak as it is further oxidised to copper(II). The experimental results agreed satisfactorily with computer calculated equilibria data using the haltafall program. The medium exchange procedure has, furthermore, been used for the determination of copper(II) in seawater reference samples, 7.5 M ammonium acetate/2.5 M acetic acid being used as stripping medium. The detection limit, after 15 min of electrolysis, was found to be 6 ng l(-1) (0.10 mM) and the relative precision 6-10%.

Determination of cadmium and lead in the low ng/l range by stripping potentiometry employing medium exchange in batch mode and multiple stripping in a hanging stripping medium drop.

A novel three-in-one electrode assembly, permitting medium exchange under a controlled potential in batch mode by exploiting electrolytic conductance through a drop hanging under the electrode, has been used for the simultaneous determination of cadmium(II) and lead(II) at trace levels. Potentiostatic reduction and amalgamation of these ions are carried out in the sample and the subsequent stripping, in the stripping potentiometry mode, in a drop of a suitable medium. Several such media have been investigated, a mixture of 2.5M acetic acid and 7.5M ammonium acetate having been found to be the most suitable with respect to sensitivity, potential resolution and trace metal purity. The quiescent conditions in the drop of medium facilitated multiple stripping and thus increased sensitivity. Detection limits for cadmium(II) and lead(II) were found to be around 0.5 ng/l. (5 and 2.5pM) for an electrolysis time of 10 min. The relative precision at the concentration level 20 ng/l. was 6.4% for cadmium and 5.4% for lead. The procedure has been used for the determination of cadmium(II) and lead(II) in reference seawater samples.

Apparent Formation of an Oxidant by Electrochemical Reduction in the Mercury(0,I,II) Chloride System.

The paradoxical appearance of a cathodic reaction sometimes observed in anodic stripping voltammetry and stripping potentiometry when using mercury film electrodes in chloride media containing mercury(II) has been investigated by systematically varying relevant chemical and electrochemical parameters and comparing the results with thermodynamic equilibrium calculations. Microscopic observations of morphological changes on the electrode surface caused by potential variations were made possible by using a novel electrode design. Three conditions have to be fulfilled for the cathodic reaction to occur: (a) formation of calomel by reaction between elemental mercury on the electrode surface and mercury(II) in solution, (b) subsequent reduction of mercury(II) to elemental mercury on the calomelized electrode surface, and (c) a chloride concentration in the range 0.001-3.5 M. Different ways of avoiding the interference from the cathodic reaction in stripping voltammetry and stripping potentiometry are experimentally demonstrated, and a mechanism for the appearance of the cathodic reaction is proposed.

Micromachined electrophoresis chips with thick-film electrochemical detectors.

A capillary electrophoresis (CE) microsystem, based on the combination of microphotolithographically fabricated separation chips and thick-film electrochemical detector strips, is described. The microsystem consists of a planar screen-printed carbon line electrode mounted perpendicular to the flow direction. Such coupling obviates the need for permanent attachment of the detector, to allow easy and fast replacement of the working electrode. Variables influencing the separation efficiency and amperometric response, including the channel-electrode spacing, separation voltage, or detection potential, are assessed and optimized. The versatility, simplicity, and low-cost advantages of the new design are coupled to an attractive performance, with submicromolar detection limits, and good precision. Applicability for assays of mixtures of nitroaromatic explosives or catecholamines is demonstrated. Such use of screen-printed detectors should also benefit conventional CE systems, particularly in applications requiring a frequent replacement of the working electrode.

Integrated electrophoresis chips/amperometric detection with sputtered gold working electrodes.

An on-chip electrochemical detector for micromachined capillary electrophoresis (CE) systems, based on sputtering a gold working electrode directly onto the capillary outlet, is described. The new on-chip detector preparation requires no microfabrication or alignment procedures nor a decoupling mechanism. The attractive performance of the integrated electrophoresis chips/amperometric detection was demonstrated for the anodic detection of neurotransmitters. The response for dopamine was linear from 20 to 200 µM, with a LOD of 1.0 µM and a sensitivity of 52 pA/µM. Such intimate coupling of capillary electrophoresis chips and electrochemical detection facilitates the realization of complete integrated microanalytical devices.

Renewable pencil electrodes for highly sensitive stripping potentiometric measurements of DNA and RNA.

Renewable graphite pencil electrodes are demonstrated to be excellent materials for adsorptive stripping measurements of trace nucleic acids. While displaying an attractive stripping performance, comparable to that of conventional carbon paste electrodes, the pencil electrode offers a convenient (mechanical) renewal, with each stripping potentiogram recorded at a fresh surface. Various pencil lead materials and lengths have been examined and experimental variables of the pretreatment and measurement procedures have been explored and optimized. The extremely low detection limits (e.g., 3 micrograms l-1 tRNA with 10 min accumulation) are coupled to a good surface-to-surface reproducibility (RSD of 6.4% for 14 repetitive measurements of 1 mg l-1 ssDNA).

Chromatographic detection of nitroaromatic and nitramine compounds by electrochemical reduction combined with photoluminescence following electron transfer.

The oxidizing agent tris(bipyridyl)ruthenium(III), or Ru-(bpy)(3)3+, is used as a postcolumn reagent for the detection of nitroaromatic and nitramine explosive compounds. After separation, the explosives are reduced electrochemically to oxidizable products such as hydroxlamines and nitrosamines, and these products react readily with Ru-(bpy)(3)3+ and Ru(bpy)(3)2+. The photoluminescence from the latter is used for detection. A porous carbon electrode was used for on-line analyte reduction following chromatography. Another porous carbon electrode was used to generate the nonluminescent Ru(bpy)(3)3+ from Ru(bpy)(3)3+ on-line at high efficiency. The two streams were combined, and the Ru(bpy)(3)2+ produced by oxidation of the reduced analytes was detected by laser illumination and light detection. Reductive hydrodynamic voltammograms of nitrobenzene, 2,4,6-trinitrotoluene, and hexahydro-1,3,5-trinitro-1,3,5-triazine indicated that a potential of - 1500 mV vs Ag/AgCl was sufficient to achieve a maximum signal from the reduced analytes. HPLC with a water/acetonitrile gradient on a C-18 reversed-phase column was then used to determine these three compounds plus the four additional examples, 1,3,5,7-tetrazocine, 2,4-dinitrotoluene; 2,6-dinitrotoluene, and 4-nitrotoluene. For both hydrodynamic voltammetry and HPLC detection, the photoluminescence following electron-transfer signal was calibrated using the one-electron standards ferrocene and ferrocenecarboxylic acid. Detection limits were in the low-nanomolar range for 20-microL injections of nonpreconcentrated nitro compounds.

Ultratrace measurements of nucleic acids by baseline-corrected adsorptive stripping square-wave voltammetry.

The direct and reliable electrochemical detection of DNA is of paramount importance to the development of modern DNA hybridization chips, for the detection of nucleic acids following their electrophoretic separations, or for the sensing of DNA damage and interactions. Such solid electrode voltammetric measurements of nucleic acids have been traditionally hampered by the large solvent decomposition background current that obscures the oxidation signals of the purine nucleobases. This paper reports on the use of adsorptive stripping square-wave voltammetry, in connection with the "moving average baseline correction" approach, for monitoring ultratrace levels of DNA and RNA. Compared to other baseline-fitted or background-subtraction protocols, the moving average baseline scheme is particularly effective in isolating the small purine nucleobase peaks, which appear as small shoulders on the steep background discharge contribution. The remarkably flat baseline thus obtained (up to extreme potentials) leads to a dramatic lowering of the detection limits to the femtomole level and to a performance that compares favorably with that of computerized chronopotentiometric measurements of nucleic acids. Combined with the speed of square-wave voltammetric measurements, such developments should expand the role of voltammetry in DNA diagnostics and nucleic acid research.

Stripping analysis of nucleic acids at a heated carbon paste electrode.

A new electrically heated carbon paste electrode has been developed for performing adsorptive stripping measurements of trace nucleic acids. Such coupling of electrochemistry at electrically heated electrodes with adsorptive constant-current stripping chronopotentiometry offers distinct advantages for trace measurements of nucleic acids. The application of increased temperatures during the deposition step results in dramatic (4-34-fold, depending on temperature applied) enhancement of the stripping signal. Such improvement is attributed to the accumulation step at the heated electrode. Forced thermal convection near the electrode surface facilitates the use of quiescent solutions and hence of ultrasmall volumes. Using an electrode temperature of 32 degrees C and a quiescent solution during the 1-min accumulation, the response is linear over the 1-8 mg/L range tested, with a detection limit of 0.5 mg/L. Such electrode heating technology offers great promise for various applications involving thermal manipulations of nucleic acids.