Next-generation sequencing and the impact on prenatal diagnosis.

INTRODUCTION: The advent of affordable and rapid next-generation sequencing has been transformative for prenatal diagnosis. Sequencing of cell-free DNA in maternal plasma has enabled the development of not only a highly sensitive screening test for fetal aneuploidies, but now definitive noninvasive prenatal diagnosis for monogenic disorders at an early gestation. Sequencing of fetal exomes offers broad diagnostic capability for pregnancies with unexpected fetal anomalies, improving the yield and accuracy of diagnoses and allowing better counseling for parents. The challenge now is to translate these approaches into mainstream use in the clinic. AREAS COVERED: Here, the authors review the current literature to describe the technologies available and how these have evolved. The opportunities and challenges at hand, including considerations for service delivery, counseling, and development of ethical guidelines, are discussed. EXPERT COMMENTARY: As technology continues to advance, future developments may be toward noninvasive fetal whole exome or whole genome sequencing and a universal method for noninvasive prenatal diagnosis without the need to sequence both parents or an affected proband. Expansion of cell-free fetal DNA analysis to include the transcriptome and the methylome is likely to yield clinical benefits for monitoring other pregnancy-related pathologies such as preeclampsia and intrauterine growth restriction

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

The role of sonographic phenotyping in delivering an efficient non-invasive prenatal diagnosis (NIPD) service for FGFR3-related skeletal dysplasias

Objectives: To evaluate the diagnostic yield of noninvasive prenatal diagnosis (NIPD) for FGFR3‐related skeletal dysplasias and assess the accuracy of referrals based on sonographic findings to inform guidelines for referral. Methods: We retrospectively reviewed laboratory and referral records from 2012 to 2018 to ascertain all NIPD tests performed using our next generation sequencing panel to detect FGFR3 mutations. We calculated the diagnostic yield of the test overall and when sub‐divided according to the phenotypic features identified on ultrasound before testing. Pregnancy outcomes were ascertained wherever possible from referring centers. Results: Of 335 tests, 261 were referred because of sonographic findings, of which 80 (31.3%) had a mutation. The diagnostic yield when short limbs were the only abnormal sonographic feature reported was 17.9% (30/168), increasing to 48.9% (23/47) in the presence of one, and 82.6% (19/23) in the presence of two or more characteristic features in addition to short limbs. Conclusions: Accurate sonographic phenotyping can maximise the diagnostic yield of NIPD in fetuses suspected to have FGFR3‐related skeletal dysplasias. We suggest that clear guidelines for referral are necessary to increase benefits, decrease costs by preventing unnecessary NIPD, and potentially allow first‐line broader spectrum testing for fetuses where the aetiology may be more heterogeneous

Associations between cardiorespiratory fitness and the metabolic syndrome in British men

Background Age and body mass index (BMI) are positively associated with the development of the metabolic syndrome (MetS). Cardiorespiratory fitness (CRF) can attenuate BMI-related increases in prevalence of MetS, but the nature of this association across different age strata has not been fully investigated. Aim To identify the association between CRF and MetS prevalence across age strata (20–69 years) and determine whether associations are independent of BMI. Methods CRF was estimated from incremental treadmill exercise in British men attending preventative health screening. Participants were placed in four age strata (20–39, 40–49, 50–59 and 60–69 years) and classified as fit or unfit using age-related cut-offs. The presence of MetS was defined using the National Cholesterol Education Program Adult Treatment Panel III criteria. Results 9666 asymptomatic men (48.7±8.4 years) were enrolled. The prevalence of MetS was 25.5% in all men and ranged from 17.1% in those aged 20–39 years to 30.6% in those aged 60–69 years. Fit men's likelihood of meeting the criteria for MetS was half (OR=0.51, 95% CI 0.46 to 0.57) that of unfit men. The likelihood of MetS was 32–53% lower across age strata in fit, compared with unfit men. Adjustment for BMI attenuated the association, though it remained significant in men aged 20–49 years. Conclusions The cardiometabolic benefits of CRF are independent of BMI particularly in men <50 years. Public health messages should emphasise the important role of CRF alongside weight management for enhancing cardiometabolic health

Delivering genome sequencing for rapid genetic diagnosis in critically ill children: parent and professional views, experiences and challenges

Rapid genomic sequencing (RGS) is increasingly being used in the care of critically ill children. Here we describe a qualitative study exploring parent and professional perspectives around the usefulness of this test, the potential for unintended harms and the challenges for delivering a wider clinical service. The Rapid Paediatric Sequencing (RaPS) study offered trio RGS for diagnosis of critically ill children with a likely monogenic disorder. Main and actionable secondary findings were reported. Semi-structured interviews were conducted with parents of children offered RGS (n = 11) and professionals (genetic clinicians, non-genetic clinicians, scientists and consenters) (n = 19) by telephone (parents n = 10/ professionals n = 1) or face-to-face (parents n = 1/professionals n = 18). We found that participants held largely positive views about RGS, describing clinical and emotional benefits from the opportunity to obtain a rapid diagnosis. Parental stress surrounding their child’s illness complicates decision making. Parental concerns are heightened when offered RGS and while waiting for results. The importance of multidisciplinary team working to enable efficient delivery of a rapid service was emphasised. Our findings give insight into the perceived value of RGS for critically ill children. Careful pre-test counselling is needed to support informed parental decision making. Many parents would benefit from additional support while waiting for results. Education of mainstream clinicians is required to facilitate clinical implementatio

Fetal central nervous system anomalies: When should we offer exome sequencing?

OBJECTIVE: To investigate the detection of pathogenic variants using exome sequencing in an international cohort of fetuses with central nervous system (CNS) anomalies. METHODS: We reviewed trio exome sequencing (ES) results for two previously reported unselected cohorts (Prenatal Assessment of Genomes and Exomes (PAGE) and CUIMC) to identify fetuses with CNS anomalies with unremarkable karyotypes and chromosomal microarrays. Variants were classified according to ACMG guidelines and association of pathogenic variants with specific types of CNS anomalies explored. RESULTS: ES was performed in 268 pregnancies with a CNS anomaly identified using prenatal ultrasound . Of those with an isolated, single, CNS anomaly, 7/97 (7.2%) had a likely pathogenic/pathogenic (LP/P) variant. This includes 3/23 (13%) fetuses with isolated mild ventriculomegaly and 3/10 (30%) fetuses with isolated agenesis of the corpus callosum. Where there were multiple anomalies within the CNS, 12/63 (19%) had LP/P variants. Of the 108 cases with CNS and other organ system anomalies, 18 (16.7%) had LP/P findings. CONCLUSION: ES is an important tool in the prenatal evaluation of fetuses with any CNS anomaly. The rate of LP/P variants tends to be highest in fetuses with multiple CNS anomalies and multisystem anomalies, however, ES may also be of benefit for isolated CNS anomalies

Noninvasive Prenatal Diagnosis for Cystic Fibrosis: Implementation, Uptake, Outcome, and Implications

BACKGROUND: Noninvasive prenatal diagnosis (NIPD) for monogenic disorders has a high uptake by families. Since 2013, our accredited public health service laboratory has offered NIPD for monogenic disorders, predominantly for de novo or paternally dominantly inherited mutations. Here we describe the extension of this service to include definitive NIPD for a recessive condition, cystic fibrosis (CF). // METHODS: Definitive NIPD for CF was developed using next-generation sequencing. Validation was performed on 13 cases from 10 families before implementation. All cases referred for CF NIPD were reviewed to determine turnaround times, genotyping results, and pregnancy outcomes. // RESULTS: Of 38 referrals, 36 received a result with a mean turnaround of 5.75 days (range, 3-11 days). Nine cases were initially inconclusive, with 3 reported unaffected because the low-risk paternal allele was inherited and 4 cases in which the high-risk paternal allele was inherited, receiving conclusive results following repeat testing. One case was inconclusive owing to a paternal recombination around the mutation site, and one case was uninformative because of no heterozygosity. Before 2016, 3 invasive referrals for CF were received annually compared with 38 for NIPD in the 24 months since offering a definitive NIPD service. // CONCLUSIONS: Timely and accurate NIPD for definitive prenatal diagnosis of CF is possible in a public health service laboratory. The method detects recombinations, and the service is well-received as evidenced by the significant increase in referrals. The bioinformatic approach is gene agnostic and will be used to expand the range of conditions tested for

Fetal exome sequencing for isolated increased nuchal translucency: should we be doing it?

Objective: To evaluate the utility of prenatal exome sequencing (ES) for isolated increased nuchal translucency (NT) and investigate factors which increase diagnostic yield. Design: Retrospective analysis of data from two prospective cohort studies. Setting: Fetal medicine centres in the UK and USA. Population: Fetuses with increased NT ≥3.5mm at 11-14 weeks’ gestation recruited to the Prenatal Assessment of Genomes and Exomes (PAGE) and Columbia fetal WES studies (n = 213). Methods: We grouped cases based on (i) the presence of additional structural abnormalities at presentation in the first trimester or later in pregnancy, and (ii) NT measurement at presentation. We compared diagnostic rates between groups using Fisher exact test. Main Outcome Measures: Detection of diagnostic genetic variants considered to have caused the observed fetal structural anomaly. Results: Diagnostic variants were detected in 12 (22.2%) of 54 fetuses presenting with non-isolated increased NT, 12 (32.4%) of 37 fetuses with isolated increased NT in the first trimester and additional abnormalities later in pregnancy, and 2 (1.8%) of 111 fetuses with isolated increased NT in the first trimester and no other abnormalities on subsequent scans. Diagnostic rate also increased with increasing size of NT. Conclusions: The diagnostic yield of prenatal ES is low for fetuses with isolated increased NT but significantly higher where there are additional structural anomalies. Prenatal ES may not be appropriate for truly isolated increased NT but timely, careful ultrasound scanning to identify other anomalies emerging later can direct testing to focus where there is a higher likelihood of diagnosis

The perseverance of Pacioli's goods inventory accounting system

This paper details sources of the 'undoubtedly strange' (Yamey, 1994a, p.119) system of goods inventory records described in Pacioli’s 1494 bookkeeping treatise and traces the longevity and widespread use of this early perpetual inventory recording (EPIR) system in English language texts. By doing so and contrasting this system with the bookkeeping treatment of modern texts, it is shown that the EPIR system persisted as the dominant form of goods inventory accounting for between 400 and 500 years and that the reasons for its demise are worthy of further consideration and research