Publications by authors named "Natalie Chandler"

14 Publications

  • Page 1 of 1

Cell-Free DNA in Pediatric Solid Organ Transplantation Using a New Detection Method of Separating Donor-Derived from Recipient Cell-Free DNA.

Clin Chem 2020 Oct;66(10):1300-1309

Department of Paediatric Nephrology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK.

Background: The use of cell-free DNA (cfDNA) as a noninvasive biomarker to detect allograft damage is expanding rapidly. However, quantifying the low fraction of donor-derived cfDNA (ddcfDNA) is challenging and requires a highly sensitive technique. ddcfDNA detection through unique donor single nucleotide polymorphisms (SNPs) is a recent new approach, however there are limited data in pediatric solid organ transplant (SOT) recipients.

Methods: We developed an assay using a combination of 61 SNPs to quantify the ddcfDNA accurately using a custom R script to model for both the patient and donor genotypes requiring only a single sample from the allograft recipient. Performance of the assay was validated using genomic DNA (gDNA), cfDNA and donor samples where available.

Results: The R "genotype-free" method gave results comparable to when using the known donor genotype. applicable to both related and unrelated pairs and can reliably measure ddcfDNA (limit of blank, below 0.12%; limit of detection, above 0.25%; limit of quantification 0.5% resulting in 84% accuracy). 159 pediatric SOT recipients (kidney, heart, and lung) were tested without the need for donor genotyping. Serial sampling was obtained from 82 patients.

Conclusion: We have developed and validated a new assay to measure the fraction of ddcfDNA in the plasma of pediatric SOT recipients. Our method can be applicable in any donor-recipient pair without the need for donor genotyping and can provide results in 48 h at a low cost. Additional prospective studies are required to demonstrate its clinical validity in a large cohort of pediatric SOT recipients.
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http://dx.doi.org/10.1093/clinchem/hvaa173DOI Listing
October 2020

The role of sonographic phenotyping in delivering an efficient noninvasive prenatal diagnosis service for FGFR3-related skeletal dysplasias.

Prenat Diagn 2020 06 25;40(7):785-791. Epub 2020 May 25.

North Thames Genomic Laboratory Hub, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom.

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.
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http://dx.doi.org/10.1002/pd.5687DOI Listing
June 2020

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

Clin Chem 2020 01;66(1):53-60

London North Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust, London, UK.

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.
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http://dx.doi.org/10.1373/clinchem.2019.304238DOI Listing
January 2020

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

Clin Chem 2020 01;66(1):207-216

NE Thames Regional Genetics Laboratories, Great Ormond Street Hospital, London, UK.

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.
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http://dx.doi.org/10.1373/clinchem.2019.305011DOI Listing
January 2020

Next-generation sequencing and the impact on prenatal diagnosis.

Expert Rev Mol Diagn 2018 08 18;18(8):689-699. Epub 2018 Jul 18.

a Genetics and Genomic Medicine , Great Ormond Street NHS Foundation Trust , London , UK.

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.
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http://dx.doi.org/10.1080/14737159.2018.1493924DOI Listing
August 2018

Prenatal diagnosis of skeletal dysplasias using a targeted skeletal gene panel.

Prenat Diagn 2018 08 3;38(9):692-699. Epub 2018 Jul 3.

Unit of Fetal Medicine, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, P.R. China.

Objective: This study aimed to perform an accurate and precise diagnosis for fetuses with suspected skeletal anomalies based on an incomplete and limited ultrasound phenotype.

Methods: Proband-only targeted skeletal gene panel sequencing was performed on 12 families who had fetuses with suspected skeletal anomalies based on ultrasound evaluations at a mean gestational age of 24 weeks and 3 days. The fetuses all had normal standard genetic testing yield (karyotyping and microarray).

Results: In 10 of 12 fetuses, panel sequencing provided a diagnosis or possible diagnosis with identification of variants in the following genes: FGFR3, COL1A2, IHH, COL2A1, and DYNC2H1. Two cases revealed novel variants in COL2A1 and DYNC2H1.

Conclusions: Our study suggests that targeted skeletal gene panel sequencing is highly sensitive for prenatal diagnosis of fetuses presenting with unexpected ultrasound findings suggestive of a skeletal dysplasia.
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http://dx.doi.org/10.1002/pd.5298DOI Listing
August 2018

Rapid prenatal diagnosis using targeted exome sequencing: a cohort study to assess feasibility and potential impact on prenatal counseling and pregnancy management.

Genet Med 2018 11 29;20(11):1430-1437. Epub 2018 Mar 29.

North Thames NHS Regional Genetics Service, Great Ormond Street NHS Foundation Trust, London, UK.

Purpose: Unexpected fetal abnormalities occur in 2-5% of pregnancies. While traditional cytogenetic and microarray approaches achieve diagnosis in around 40% of cases, lack of diagnosis in others impedes parental counseling, informed decision making, and pregnancy management. Postnatally exome sequencing yields high diagnostic rates, but relies on careful phenotyping to interpret genotype results. Here we used a multidisciplinary approach to explore the utility of rapid fetal exome sequencing for prenatal diagnosis using skeletal dysplasias as an exemplar.

Methods: Parents in pregnancies undergoing invasive testing because of sonographic fetal abnormalities, where multidisciplinary review considered skeletal dysplasia a likely etiology, were consented for exome trio sequencing (both parents and fetus). Variant interpretation focused on a virtual panel of 240 genes known to cause skeletal dysplasias.

Results: Definitive molecular diagnosis was made in 13/16 (81%) cases. In some cases, fetal ultrasound findings alone were of sufficient severity for parents to opt for termination. In others, molecular diagnosis informed accurate prediction of outcome, improved parental counseling, and enabled parents to terminate or continue the pregnancy with certainty.

Conclusion: Trio sequencing with expert multidisciplinary review for case selection and data interpretation yields timely, high diagnostic rates in fetuses presenting with unexpected skeletal abnormalities. This improves parental counseling and pregnancy management.
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http://dx.doi.org/10.1038/gim.2018.30DOI Listing
November 2018

Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect.

Proc Natl Acad Sci U S A 2016 Mar 16;113(9):E1236-45. Epub 2016 Feb 16.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom;

Xeroderma pigmentosum (XP) is a rare DNA repair disorder characterized by increased susceptibility to UV radiation (UVR)-induced skin pigmentation, skin cancers, ocular surface disease, and, in some patients, sunburn and neurological degeneration. Genetically, it is assigned to eight complementation groups (XP-A to -G and variant). For the last 5 y, the UK national multidisciplinary XP service has provided follow-up for 89 XP patients, representing most of the XP patients in the United Kingdom. Causative mutations, DNA repair levels, and more than 60 clinical variables relating to dermatology, ophthalmology, and neurology have been measured, using scoring systems to categorize disease severity. This deep phenotyping has revealed unanticipated heterogeneity of clinical features, between and within complementation groups. Skin cancer is most common in XP-C, XP-E, and XP-V patients, previously considered to be the milder groups based on cellular analyses. These patients have normal sunburn reactions and are therefore diagnosed later and are less likely to adhere to UVR protection. XP-C patients are specifically hypersensitive to ocular damage, and XP-F and XP-G patients appear to be much less susceptible to skin cancer than other XP groups. Within XP groups, different mutations confer susceptibility or resistance to neurological damage. Our findings on this large cohort of XP patients under long-term follow-up reveal that XP is more heterogeneous than has previously been appreciated. Our data now enable provision of personalized prognostic information and management advice for each XP patient, as well as providing new insights into the functions of the XP proteins.
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http://dx.doi.org/10.1073/pnas.1519444113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780618PMC
March 2016

A Distinct Genotype of XP Complementation Group A: Surprisingly Mild Phenotype Highly Prevalent in Northern India/Pakistan/Afghanistan.

J Invest Dermatol 2016 Apr 29;136(4):869-872. Epub 2015 Dec 29.

National Xeroderma Pigmentosum Service, Department of Photodermatology, St. John's Institute of Dermatology, Guy's and St. Thomas' NHS Foundation Trust, London, United Kingdom. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2015.12.031DOI Listing
April 2016

Computer three-dimensional anatomical reconstruction of the human sinus node and a novel paranodal area.

Anat Rec (Hoboken) 2011 Jun 28;294(6):970-9. Epub 2011 Apr 28.

Cardiovascular Medicine, University of Manchester, Manchester, UK.

We have previously shown in rabbit that the pacemaker of the heart (the sinus node) is widespread and matches the wide distribution of the leading pacemaker site within the right atrium. There is, however, uncertainty about the precise location of the pacemaker in human heart, and its spatial relationships with the surrounding right atrial muscle. Therefore, the aim of the current study was to investigate the distribution of the sinus node tissue in a series of healthy human hearts and, for one of the hearts to construct a computer three-dimensional anatomical model of the sinus node, including the likely orientation of myocytes. A combination of experimental techniques--diffusion tensor magnetic resonance imaging (DT-MRI), histology, immunohistochemistry, image processing and computer modelling--was used. Our data show that the sinus node was larger than in previous studies and, most importantly, we identified a previously unknown area running alongside the sinus node (the "paranodal area"), which is even more extensive than the sinus node. This area possesses properties of both nodal and atrial tissues and may have a role in pacemaking. For example, it could explain the wide spread distribution of the leading pacemaker site in human right atrium, a phenomenon known as the wandering pacemaker observed in clinics. In summary, a novel 3D anatomical reconstruction presents a new picture of the distribution of nodal cells within the human right atrium.
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http://dx.doi.org/10.1002/ar.21379DOI Listing
June 2011

Hysteresis in human HCN4 channels: a crucial feature potentially affecting sinoatrial node pacemaking.

Sheng Li Xue Bao 2010 Feb;62(1):1-13

Cardiac Rhythm Disease Management, Medtronic Inc., Mounds View, MN 55112, USA.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels modulate and regulate cardiac rhythm and rate. It has been suggested that, unlike the HCN1 and HCN2 channels, the slower HCN4 channel may not exhibit voltage-dependent hysteresis. We studied the electrophysiological properties of human HCN4 (hHCN4) channels and its modulation by cAMP to determine whether hHCN4 exhibits hysteresis, by using single-cell patch-clamp in HEK293 cells stably transfected with hHCN4. Quantitative real-time RT-PCR was also used to determine levels of expression of HCNs in human cardiac tissue. Voltage-clamp analysis revealed that hHCN4 current (I(h)) activation shifted in the depolarizing direction with more hyperpolarized holding potentials. Triangular ramp and action potential clamp protocols also revealed hHCN4 hysteresis. cAMP enhanced I(h) and shifted activation in the depolarizing direction, thus modifying the intrinsic hHCN4 hysteresis behavior. Quantitative PCR analysis of human sinoatrial node (SAN) tissue showed that HCN4 accounts for 75% of the HCNs in human SAN while HCN1 (21%), HCN2 (3%), and HCN3 (0.7%) constitute the remainder. Our data suggest that HCN4 is the predominant HCN subtype in the human SAN and that I(h) exhibits voltage-dependent hysteresis behavior that can be modified by cAMP. Therefore, hHCN4 hysteresis potentially plays a crucial role in human SAN pacemaking activity.
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February 2010

Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker.

Circulation 2009 Mar 16;119(12):1562-75. Epub 2009 Mar 16.

Cardiovascular Research Group, Faculty of Medical and Human Sciences, University of Manchester, Core Technology Facility, 46 Grafton St, Manchester M139NT, United Kingdom.

Background: Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity.

Methods And Results: Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na(v)1.5, K(v)4.3, K(v)1.5, ERG, K(ir)2.1, K(ir)6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca(v)1.3, Ca(v)3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K(v)4.2, K(ir)6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking.

Conclusions: Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.108.804369DOI Listing
March 2009

P2 purinergic receptor mRNA in rat and human sinoatrial node and other heart regions.

Naunyn Schmiedebergs Arch Pharmacol 2009 Jun 21;379(6):541-9. Epub 2009 Feb 21.

Cardiovascular Research Group, Faculty of Medical and Human Sciences, University of Manchester, Core Technology Facility, 46 Grafton Street, Manchester, M13 9NT, UK.

It is known that adenosine 5'-triphosphate (ATP) is a cotransmitter in the heart. Additionally, ATP is released from ischemic and hypoxic myocytes. Therefore, cardiac-derived sources of ATP have the potential to modify cardiac function. ATP activates P2X(1-7) and P2Y(1-14) receptors; however, the presence of P2X and P2Y receptor subtypes in strategic cardiac locations such as the sinoatrial node has not been determined. An understanding of P2X and P2Y receptor localization would facilitate investigation of purine receptor function in the heart. Therefore, we used quantitative PCR and in situ hybridization to measure the expression of mRNA of all known purine receptors in rat left ventricle, right atrium and sinoatrial node (SAN), and human right atrium and SAN. Expression of mRNA for all the cloned P2 receptors was observed in the ventricles, atria, and SAN of the rat. However, their abundance varied in different regions of the heart. P2X(5) was the most abundant of the P2X receptors in all three regions of the rat heart. In rat left ventricle, P2Y(1), P2Y(2), and P2Y(14) mRNA levels were highest for P2Y receptors, while in right atrium and SAN, P2Y(2) and P2Y(14) levels were highest, respectively. We extended these studies to investigate P2X(4) receptor mRNA in heart from rats with coronary artery ligation-induced heart failure. P2X(4) receptor mRNA was upregulated by 93% in SAN (P < 0.05), while a trend towards an increase was also observed in the right atrium and left ventricle (not significant). Thus, P2X(4)-mediated effects might be modulated in heart failure. mRNA for P2X(4-7) and P2Y(1,2,4,6,12-14), but not P2X(2,3) and P2Y(11), was detected in human right atrium and SAN. In addition, mRNA for P2X(1) was detected in human SAN but not human right atrium. In human right atrium and SAN, P2X(4) and P2X(7) mRNA was the highest for P2X receptors. P2Y(1) and P2Y(2) mRNA were the most abundant for P2Y receptors in the right atrium, while P2Y(1), P2Y(2), and P2Y(14) were the most abundant P2Y receptor subtypes in human SAN. This study shows a widespread distribution of P2 receptor mRNA in rat heart tissues but a more restricted presence and distribution of P2 receptor mRNA in human atrium and SAN. This study provides further direction for the elucidation of P2 receptor modulation of heart rate and contractility.
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http://dx.doi.org/10.1007/s00210-009-0403-2DOI Listing
June 2009

The anatomy of the cardiac conduction system.

Clin Anat 2009 Jan;22(1):99-113

Cardiovascular Research Group, Faculty of Medical and Human Sciences, University of Manchester, Core Technology Facility, 46 Grafton Street, Manchester, United Kingdom.

All the myocytes within the heart have the capacity to conduct the cardiac impulse. A population of myocytes is specialized so as to generate the cardiac impulse and then to conduct it from the atrial to the ventricular chambers. This population has become known as the conduction system. Anatomists who seek to demonstrate the location of the components of this system must contend with the fact that the components of the system cannot be distinguished from the working myocardial elements by gross dissection. In important presentations to the German Pathological Society in 1910, rules were suggested for the histological distinction of these conducting cells. These rules proposed that the myocytes, to be considered as part of the conduction system, should be histologically discrete, traceable from section to section in serially prepared material, and if to be considered as tracts, should be insulated by fibrous tissue from the adjacent myocytes. Immunohistochemical techniques have now been developed that better demonstrate the distinction between the cells specialized to conduct from working myocytes. These new techniques, for the most part, confirm the accuracy of the initial descriptions. They also reveal additional areas with the characteristics of conduction tissues. These additional areas are located in a paranodal area adjacent to the sinus node, in the vestibules of both atrioventricular valvar orifices, and in a partial ring around the aortic root. In this review, we describe all these features, emphasizing the relationship of the newly recognized components to the established parts of the cardiac conduction system, and how the new findings need to be assessed in the light of the old criteria.
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http://dx.doi.org/10.1002/ca.20700DOI Listing
January 2009