Developing variant interpretation pipelines for inherited retinal diseases and ciliopathies: using medical genomics to improve diagnostic yield

Primary ciliopathies are a group of rare inherited disorders caused by defects in the structure or function of primary cilia (the ‘cell’s antenna’). This thesis describes approaches to improve molecular diagnosis rates for primary ciliopathy patients over the ~40-80% currently achieved, through whole genome sequencing (WGS) analysis and functional variant interpretation. Firstly, I analysed WGS data from the 100,000 Genomes Project (100K) for participants who were clinically suspected to have primary ciliopathies. I identified a molecular diagnosis rate for n=45/83 (54.2%), providing a 21.7% diagnostic uplift compared to results previously reported by Genomics England (GEL). I then performed a reverse phenotyping study, starting by looking for pathogenic variants in nine multisystemic ciliopathy disease genes across the 100K rare disease dataset. This was linked back to available clinical data, aiming to identify participants with “hidden” ciliopathy diagnoses recruited to alternative categories. I identified 18 new, reportable diagnoses and 44 previously reported by GEL. I also found 11 un-reportable molecular diagnoses, lacking key clinical features to provide a confident fit for phenotype. This shows that the quality of entered phenotypic data is critical to allow accurate genotype-phenotype correlation. In a third study, I developed strategies for functional interpretation of eight TMEM67 missense variants of uncertain significance (VUSs) with collaborators in Ireland, using CRISPR/Cas9 gene editing in a human ciliated cell-line (RPE-1) and C. elegans. These assays provided interpretation of three VUS as benign and five as pathogenic. The two 100K studies show that diagnosis rates for ciliopathies can be improved through WGS analysis, especially structural and splice variant analysis. We are a long way from delivering a high-throughput system for VUS interpretation that could provide clinical utility in the diagnostic setting. Overall, we have provided benefit for ciliopathy patients through additional molecular diagnoses, accompanied by transferable skills applicable to wider patient group

The impact of fibroid characteristics on pregnancy outcome

Objective The objective of the study was to assess the influence of different characteristics of fibroids on pregnancy outcome. Study Design We identified women with fibroids 4 cm or greater in size on ultrasonography at the dating scan between January 2002 and December 2012. The size (4-7 cm, 7-10 cm, >10 cm), number (multiple/single), location (lower uterus/body of uterus), and type (intramural, combination of intramural/subserosal, subserosal) were ascertained. Medical records were reviewed to obtain pregnancy outcomes (preterm delivery, birthweight, mode of delivery, estimated blood loss, postpartum hemorrhage, and admission for fibroid-related pain)

Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study

Background: Fetal structural anomalies, which are detected by ultrasonography, have a range of genetic causes, including chromosomal aneuploidy, copy number variations (CNVs; which are detectable by chromosomal microarrays), and pathogenic sequence variants in developmental genes. Testing for aneuploidy and CNVs is routine during the investigation of fetal structural anomalies, but there is little information on the clinical usefulness of genome-wide next-generation sequencing in the prenatal setting. We therefore aimed to evaluate the proportion of fetuses with structural abnormalities that had identifiable variants in genes associated with developmental disorders when assessed with whole-exome sequencing (WES). Methods: In this prospective cohort study, two groups in Birmingham and London recruited patients from 34 fetal medicine units in England and Scotland. We used whole-exome sequencing (WES) to evaluate the presence of genetic variants in developmental disorder genes (diagnostic genetic variants) in a cohort of fetuses with structural anomalies and samples from their parents, after exclusion of aneuploidy and large CNVs. Women were eligible for inclusion if they were undergoing invasive testing for identified nuchal translucency or structural anomalies in their fetus, as detected by ultrasound after 11 weeks of gestation. The partners of these women also had to consent to participate. Sequencing results were interpreted with a targeted virtual gene panel for developmental disorders that comprised 1628 genes. Genetic results related to fetal structural anomaly phenotypes were then validated and reported postnatally. The primary endpoint, which was assessed in all fetuses, was the detection of diagnostic genetic variants considered to have caused the fetal developmental anomaly. Findings: The cohort was recruited between Oct 22, 2014, and June 29, 2017, and clinical data were collected until March 31, 2018. After exclusion of fetuses with aneuploidy and CNVs, 610 fetuses with structural anomalies and 1202 matched parental samples (analysed as 596 fetus-parental trios, including two sets of twins, and 14 fetus-parent dyads) were analysed by WES. After bioinformatic filtering and prioritisation according to allele frequency and effect on protein and inheritance pattern, 321 genetic variants (representing 255 potential diagnoses) were selected as potentially pathogenic genetic variants (diagnostic genetic variants), and these variants were reviewed by a multidisciplinary clinical review panel. A diagnostic genetic variant was identified in 52 (8·5%; 95% CI 6·4–11·0) of 610 fetuses assessed and an additional 24 (3·9%) fetuses had a variant of uncertain significance that had potential clinical usefulness. Detection of diagnostic genetic variants enabled us to distinguish between syndromic and non-syndromic fetal anomalies (eg, congenital heart disease only vs a syndrome with congenital heart disease and learning disability). Diagnostic genetic variants were present in 22 (15·4%) of 143 fetuses with multisystem anomalies (ie, more than one fetal structural anomaly), nine (11·1%) of 81 fetuses with cardiac anomalies, and ten (15·4%) of 65 fetuses with skeletal anomalies; these phenotypes were most commonly associated with diagnostic variants. However, diagnostic genetic variants were least common in fetuses with isolated increased nuchal translucency (≥4·0 mm) in the first trimester (in three [3·2%] of 93 fetuses). Interpretation: WES facilitates genetic diagnosis of fetal structural anomalies, which enables more accurate predictions of fetal prognosis and risk of recurrence in future pregnancies. However, the overall detection of diagnostic genetic variants in a prospectively ascertained cohort with a broad range of fetal structural anomalies is lower than that suggested by previous smaller-scale studies of fewer phenotypes. WES improved the identification of genetic disorders in fetuses with structural abnormalities; however, before clinical implementation, careful consideration should be given to case selection to maximise clinical usefulness.The PAGE study is supported by a Health Innovation Challenge from the UK Department of Health and Wellcome Trust (no. HICF-R7-396). Additionally, LSC is partially funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at Great Ormond Street Hospital and ERM acknowledges support from NIHR Cambridge Biomedical Research Centre (an NIHR Senior Investigator Award). The University of Cambridge has received salary support with regard to ERM from the UK National Health Service (NHS) in the east of England through the Clinical Academic Reserve

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

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.Genetics in Medicine advance online publication, 29 March 2018; doi:10.1038/gim.2018.30

Clinical features and management of children with primary ciliary dyskinesia in England

Objective In England, the National Health Service commissioned a National Management Service for children with primary ciliary dyskinesia (PCD). The aims of this study were to describe the health of children seen in this Service and compare lung function to children with cystic fibrosis (CF). Design Multi-centre service evaluation of the English National Management PCD Service. Setting Four nationally commissioned PCD centres in England. Patients 333 children with PCD reviewed in the Service in 2015; lung function data were also compared to 2970 children with CF. Results Median age at diagnosis for PCD was 2.6 years, significantly lower in children with situs inversus (1.0 vs 6.0 years, p<0.0001). Compared with national data from the CF Registry, mean (SD) %predicted FEV1 76.8% in PCD (n=240) and 85.0% in CF, and FEV1 was lower in children with PCD up to the age of 15 years. Approximately half of children had some hearing impairment, with 26% requiring hearing aids. Children with a lower BMI had lower FEV1 (p<0.001). One third of children had positive respiratory cultures at review, 54% of these grew Haemophilus influenzae. Conclusions We provide evidence that children with PCD in England have worse lung function than those with CF. Nutritional status should be considered in PCD management, as those with a lower BMI have significantly lower FEV1. Hearing impairment is common but seems to improve with age. Well designed and powered randomised controlled trials on management of PCD are needed to inform best clinical practice

Risk factors for situs defects and congenital heart disease in primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is associated with abnormal organ positioning (situs) and congenital heart disease (CHD). This study investigated genotype–phenotype associations in PCD to facilitate risk predictions for cardiac and laterality defects. This retrospective cohort study of 389 UK patients with PCD found 51% had abnormal situs and 25% had CHD and/or laterality defects other than situs inversus totalis. Patients with biallelic mutations in a subset of nine PCD genes had normal situs. Patients with consanguineous parents had higher odds of situs abnormalities than patients with non-consanguineous parents. Patients with abnormal situs had higher odds of CHD and/or laterality defects

Molecular diagnoses in the congenital malformations caused by ciliopathies cohort of the 100,000 Genomes Project

Background: Primary ciliopathies represent a group of inherited disorders due to defects in the primary cilium, the ‘cell’s antenna’. The 100,000 Genomes Project was launched in 2012 by Genomics England (GEL), recruiting National Health Service (NHS) patients with eligible rare diseases and cancer. Sequence data were linked to Human Phenotype Ontology (HPO) terms entered by recruiting clinicians.Methods: Eighty-three prescreened probands were recruited to the 100,000 Genomes Project suspected to have congenital malformations caused by ciliopathies in the following disease categories: Bardet-Biedl syndrome (n=45), Joubert syndrome (n=14) and ‘Rare Multisystem Ciliopathy Disorders’ (n=24). We implemented a bespoke variant filtering and analysis strategy to improve molecular diagnostic rates for these participants.Results: We determined a research molecular diagnosis for n=43/83 (51.8%) probands. This is 19.3% higher than previously reported by GEL (n=27/83 (32.5%)). A high proportion of diagnoses are due to variants in non-ciliopathy disease genes (n=19/43, 44.2%) which may reflect difficulties in clinical recognition of ciliopathies. n=11/83 probands (13.3%) had at least one causative variant outside the tiers 1 and 2 variant prioritisation categories (GEL’s automated triaging procedure), which would not be reviewed in standard 100,000 Genomes Project diagnostic strategies. These include four structural variants and three predicted to cause non-canonical splicing defects. Two unrelated participants have biallelic likely pathogenic variants in LRRC45, a putative novel ciliopathy disease gene.Conclusion: These data illustrate the power of linking large-scale genome sequence to phenotype information. They demonstrate the value of research collaborations in order to maximise interpretation of genomic data