Non-invasive prenatal testing for aneuploidy, copy number variants and single gene disorders

The discovery of cell-free fetal DNA (cffDNA) in maternal plasma has enabled a paradigm shift in prenatal testing, allowing for safer, earlier detection of genetic conditions of the fetus. Non-invasive prenatal testing (NIPT) for fetal aneuploidies has provided an alternative, highly efficient approach to first-trimester aneuploidy screening, and since its inception has been rapidly adopted worldwide. Due to the genome-wide nature of some NIPT protocols, the commercial sector has widened the scope of cell-free DNA (cfDNA) screening to include sex chromosome aneuploidies, rare autosomal trisomies and sub-microscopic copy-number variants. These developments may be marketed as ‘expanded NIPT’ or ‘NIPT Plus’ and bring with them a plethora of ethical and practical considerations. Concurrently, cfDNA tests for single-gene disorders, termed non-invasive prenatal diagnosis (NIPD), have been developed for an increasing array of conditions but are less widely available. Despite the fact that all these tests utilise the same biomarker, cfDNA, there is considerable variation in key parameters such as sensitivity, specificity and positive predictive value depending on what the test is for. The distinction between diagnostics and screening has become blurred, and there is a clear need for the education of physicians and patients regarding the technical capabilities and limitations of these different forms of testing. Furthermore, there is a requirement for consistent guidelines that apply across health sectors, both public and commercial, to ensure that tests are validated and robust and that careful and appropriate pre-test and post-test counselling is provided by professionals who understand the tests offered

Noninvasive Prenatal Diagnosis of Single-Gene Diseases: The Next Frontier

BACKGROUND: Cell-free fetal DNA (cffDNA) is present in the maternal blood from around 4 weeks gestation and makes up 5%-20% of the total circulating cell-free DNA (cfDNA) in maternal plasma. Presence of cffDNA has allowed development of noninvasive prenatal diagnosis (NIPD) for single-gene disorders. This can be performed from 9 weeks gestation and offers a definitive diagnosis without the miscarriage risk associated with invasive procedures. One of the major challenges is distinguishing fetal mutations in the high background of maternal cfDNA, and research is currently focusing on the technological advances required to solve this problem. CONTENT: Here, we review the literature to describe the current status of NIPD for monogenic disorders and discuss how the evolving methodologies and technologies are expected to impact this field in both the commercial and public healthcare setting. SUMMARY: NIPD for single-gene diseases was first reported in 2000 and took 12 years to be approved for use in a public health service. Implementation has remained slow but is expected to increase as this testing becomes cheaper, faster, and more accurate. There are still many technical and analytical challenges ahead, and it is vital that discussions surrounding the ethical and social impact of NIPD take account of the considerations required to implement these services safely into the healthcare setting, while keeping up with the technological advances

Enhanced mitochondrial genome analysis: bioinformatic and long-read sequencing advances and their diagnostic implications

Introduction: Primary mitochondrial diseases (PMDs) comprise a large and heterogeneous group of genetic diseases that result from pathogenic variants in either nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Widespread adoption of next-generation sequencing (NGS) has improved the efficiency and accuracy of mtDNA diagnoses; however, several challenges remain. Areas covered: In this review, we briefly summarize the current state of the art in molecular diagnostics for mtDNA and consider the implications of improved whole genome sequencing (WGS), bioinformatic techniques, and the adoption of long-read sequencing, for PMD diagnostics. Expert opinion: We anticipate that the application of PCR-free WGS from blood DNA will increase in diagnostic laboratories, while for adults with myopathic presentations, WGS from muscle DNA may become more widespread. Improved bioinformatic strategies will enhance WGS data interrogation, with more accurate delineation of mtDNA and NUMTs (nuclear mitochondrial DNA segments) in WGS data, superior coverage uniformity, indirect measurement of mtDNA copy number, and more accurate interpretation of heteroplasmic large-scale rearrangements (LSRs). Separately, the adoption of diagnostic long-read sequencing could offer greater resolution of complex LSRs and the opportunity to phase heteroplasmic variants

Formation of bitumen in the Elgin/Franklin complex, Central Graben, North Sea: Implications for hydrocarbon charging

The Elgin/Franklin complex contains gas condensates in Upper Jurassic reservoirs in the North Sea Central Graben. Upper parts of the reservoirs contain bitumens, which previous studies have suggested were formed by the thermal cracking of oil as the reservoirs experienced temperatures >150°C during rapid Plio-Pleistocene subsidence. Bitumen-stained cores contaminated by oil-based drilling muds have been analysed by hydropyrolysis. Asphaltene-bound aliphatic hydrocarbon fractions were dominated by n-hexadecane and n-octadecane originating from fatty acid additives in the muds. Uncontaminated asphaltene-bound aromatic hydrocarbon fractions however contained a PAH distribution very similar to normal North Sea oils, suggesting that the bitumens may not have been derived from oil cracking.1-D basin models of well 29/5b-6 and a pseudowell east of the Elgin/Franklin complex utilise a thermal history derived from the basin’s rifting and subsidence histories, combined with the conservation of energy currently not contained in the thermal histories. Vitrinite reflectance values predicted by the conventional kinetic models do not match the measured data. Using the pressure-dependent PresRo® model, however, a good match was achieved between observed and measured data. The predicted petroleum generation is combined with published diagenetic cement data from Elgin/Franklin to produce a composite model for petroleum generation, diagenetic-cement and bitumen formation

Impact of high water pressure on oil generation and maturation in Kimmeridge Clay and Monterey source rocks: implications for petroleum retention and gas generation in shale gas systems

This study presents results for pyrolysis experiments conducted on immature Type II and IIs source rocks (Kimmeridge Clay, Dorset UK, and Monterey shale, California, USA respectively) to investigate the impact of high water pressure on source rock maturation and petroleum (oil and gas) generation. Using a 25 ml Hastalloy vessel, the source rocks were pyrolysed at low (180 and 245 bar) and high (500, 700 and 900 bar) water pressure hydrous conditions at 350 °C and 380 °C for between 6 and 24 h. For the Kimmeridge Clay (KCF) at 350 °C, Rock Eval HI of the pyrolysed rock residues were 30–44 mg/g higher between 6 h and 12 h at 900 bar than at 180 bar. Also at 350 °C for 24 h the gas, expelled oil, and vitrinite reflectance (VR) were all reduced by 46%, 61%, and 0.25% Ro respectively at 900 bar compared with 180 bar. At 380 °C the retardation effect of pressure on the KCF was less significant for gas generation. However, oil yield and VR were reduced by 47% and 0.3% Ro respectively, and Rock Eval HI was also higher by 28 mg/g at 900 bar compared with 245 bar at 12 h. The huge decrease in gas and oil yields and the VR observed with an increase in water pressure at 350 °C for 24 h and 380 °C for 12 h (maximum oil generation) were also observed for all other times and temperatures investigated for the KCF and the Monterey shale. This shows that high water pressure significantly retards petroleum generation and source rock maturation. The retardation of oil generation and expulsion resulted in significant amounts of bitumen and oil being retained in the rocks pyrolysed at high pressures, suggesting that pressure is a possible mechanism for retaining petroleum (bitumen and oil) in source rocks. This retention of petroleum within the rock provides a mechanism for oil-prone source rocks to become potential shale gas reservoirs. The implications from this study are that in geological basins, pressure, temperature and time will all exert significant control on the extent of petroleum generation and source rock maturation for Type II source rocks, and that the petroleum retained in the rocks at high pressures may explain in part why oil-prone source rocks contain the most prolific shale gas resources

Non-invasive fetal genotyping for maternal alleles with droplet digital PCR: A comparative study of analytical approaches

This is the final version. Available on open access from Wiley via the DOI in this recordData availability statement: The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.OBJECTIVES: To develop a flexible droplet digital PCR (ddPCR) workflow to perform non-invasive prenatal diagnosis via relative mutation dosage (RMD) for maternal pathogenic variants with a range of inheritance patterns, and to compare the accuracy of multiple analytical approaches. METHODS: Cell free DNA (cfDNA) was tested from 124 archived maternal plasma samples: 88 cases for sickle cell disease and 36 for rare Mendelian conditions. Three analytical methods were compared: sequential probability ratio testing (SPRT), Bayesian and z-score analyses. RESULTS: The SPRT, Bayesian and z-score analyses performed similarly well with correct prediction rates of 96%, 97% and 98%, respectively. However, there were high rates of inconclusive results for each cohort, particularly for z-score analysis which was 31% overall. Two samples were incorrectly classified by all three analytical methods; a false negative result predicted for a fetus affected with sickle cell disease and a false positive result predicting the presence of an X-linked IDS variant in an unaffected fetus. CONCLUSIONS: ddPCR can be applied to RMD for diverse conditions and inheritance patterns, but all methods carry a small risk of erroneous results. Further evaluation is required both to reduce the rate of inconclusive results and explore discordant results in more detail.National Institute for Health and Care Research (NIHR

Evaluation of the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in infantile epilepsy (Gene-STEPS): an international, multicentre, pilot cohort study

BACKGROUND: Most neonatal and infantile-onset epilepsies have presumed genetic aetiologies, and early genetic diagnoses have the potential to inform clinical management and improve outcomes. We therefore aimed to determine the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in this population. METHODS: We conducted an international, multicentre, cohort study (Gene-STEPS), which is a pilot study of the International Precision Child Health Partnership (IPCHiP). IPCHiP is a consortium of four paediatric centres with tertiary-level subspecialty services in Australia, Canada, the UK, and the USA. We recruited infants with new-onset epilepsy or complex febrile seizures from IPCHiP centres, who were younger than 12 months at seizure onset. We excluded infants with simple febrile seizures, acute provoked seizures, known acquired cause, or known genetic cause. Blood samples were collected from probands and available biological parents. Clinical data were collected from medical records, treating clinicians, and parents. Trio genome sequencing was done when both parents were available, and duo or singleton genome sequencing was done when one or neither parent was available. Site-specific protocols were used for DNA extraction and library preparation. Rapid genome sequencing and analysis was done at clinically accredited laboratories, and results were returned to families. We analysed summary statistics for cohort demographic and clinical characteristics and the timing, diagnostic yield, and clinical impact of rapid genome sequencing. FINDINGS: Between Sept 1, 2021, and Aug 31, 2022, we enrolled 100 infants with new-onset epilepsy, of whom 41 (41%) were girls and 59 (59%) were boys. Median age of seizure onset was 128 days (IQR 46-192). For 43 (43% [binomial distribution 95% CI 33-53]) of 100 infants, we identified genetic diagnoses, with a median time from seizure onset to rapid genome sequencing result of 37 days (IQR 25-59). Genetic diagnosis was associated with neonatal seizure onset versus infantile seizure onset (14 [74%] of 19 vs 29 [36%] of 81; p=0·0027), referral setting (12 [71%] of 17 for intensive care, 19 [44%] of 43 non-intensive care inpatient, and 12 [28%] of 40 outpatient; p=0·0178), and epilepsy syndrome (13 [87%] of 15 for self-limited epilepsies, 18 [35%] of 51 for developmental and epileptic encephalopathies, 12 [35%] of 34 for other syndromes; p=0·001). Rapid genome sequencing revealed genetic heterogeneity, with 34 unique genes or genomic regions implicated. Genetic diagnoses had immediate clinical utility, informing treatment (24 [56%] of 43), additional evaluation (28 [65%]), prognosis (37 [86%]), and recurrence risk counselling (all cases). INTERPRETATION: Our findings support the feasibility of implementation of rapid genome sequencing in the clinical care of infants with new-onset epilepsy. Longitudinal follow-up is needed to further assess the role of rapid genetic diagnosis in improving clinical, quality-of-life, and economic outcomes. FUNDING: American Academy of Pediatrics, Boston Children's Hospital Children's Rare Disease Cohorts Initiative, Canadian Institutes of Health Research, Epilepsy Canada, Feiga Bresver Academic Foundation, Great Ormond Street Hospital Charity, Medical Research Council, Murdoch Children's Research Institute, National Institute of Child Health and Human Development, National Institute for Health and Care Research Great Ormond Street Hospital Biomedical Research Centre, One8 Foundation, Ontario Brain Institute, Robinson Family Initiative for Transformational Research, The Royal Children's Hospital Foundation, University of Toronto McLaughlin Centre

The effect of water pressure on hydrocarbon generation reactions : some inferences from laboratory experiments

For the last twenty-five years most petroleum geochemists and basin modellers have produced and used models for maturation and hydrocarbon generation reactions in geological basins that do not consider pressure as a primary control. These conclusions are based on extensive laboratory investigations mainly using pyrolysis. Chemical theory, however, indicates that endothermic volume expansion reactions, such as maturation and hydrocarbon generation, are controlled by both the system pressure and temperature, and geochemists and basin modellers may need to reconsider the importance of pressure on maturation and hydrocarbon generation reactions in geological basins. Unusual earlier studies (at least in terms of petroleum geochemical pyrolysis research) used a vessel in which the pressure is entirely derived from liquid water rather than both liquid water and vapour, as in hydrous pyrolysis experimentation. Results from these experiments showed that both total organic carbon (TOC) and hydrogen index (HI) were elevated in the pyrolysed kerogen residue, suggesting that hydrocarbon generation was being retarded by the effect of water pressure. This paper presents the results of an experimental investigation into the effects of water pressure and phase on hydrocarbon generation and expulsion from the Kimmeridge Clay Formation (KCF) in the temperature range 310–350°C and in the pressure range 0–500 bar, and presents quantitative results both for the amounts of gas and bitumen generated and the composition of the generated gas. The experimental results show that the water pressure retards both bitumen and gas generation, with gas generation being retarded more severely than bitumen generation