Christopher Boustred

Improving diagnosis and broadening the phenotypes in early-onset seizure and severe developmental delay disorders through gene panel analysis

Background We sought to investigate the diagnostic yield and mutation spectrum in previously reported genes for early-onset epilepsy and disorders of severe developmental delay. Methods In 400 patients with these disorders with no known underlying aetiology and no major structural brain anomaly, we analysed 46 genes using a combination of targeted sequencing on an Illumina MiSeq platform and targeted, exon-level microarray copy number analysis. Results We identified causative mutations in 71/400 patients (18%). The diagnostic rate was highest among those with seizure onset within the first two months of life (39%), although overall it was similar in those with and without seizures. The most frequently mutated gene was SCN2A (11 patients, 3%). Other recurrently mutated genes included CDKL5, KCNQ2, SCN8A (six patients each), FOXG1, MECP2, SCN1A, STXBP1 (five patients each), KCNT1, PCDH19, TCF4 (three patients each) and ATP1A3, PRRT2 and SLC9A6 (two patients each). Mutations in EHMT1, GABRB3, LGI1, MBD5, PIGA, UBE3A and ZEB2 were each found in single patients. We found mutations in a number of genes in patients where either the electroclinical features or dysmorphic phenotypes were atypical for the identified gene. In only 11 cases (15%) had the clinician sufficient certainty to specify the mutated gene as the likely cause before testing. Conclusions Our data demonstrate the considerable utility of a gene panel approach in the diagnosis of patients with early-onset epilepsy and severe developmental delay disorders., They provide further insights into the phenotypic spectrum and genotype–phenotype correlations for a number of the causative genes and emphasise the value of exon-level copy number testing in their analysis.

Exome sequencing for prenatal diagnosis of fetuses with sonographic abnormalities

In the absence of aneuploidy or other pathogenic cytogenetic abnormality, fetuses with increased nuchal translucency (NT ≥ 3.5 mm) and/or other sonographic abnormalities have a greater incidence of genetic syndromes, but defining the underlying pathology can be challenging. Here, we investigate the value of whole exome sequencing in fetuses with sonographic abnormalities but normal microarray analysis.

Evaluation of non-invasive prenatal testing (NIPT) for aneuploidy in an NHS setting: a reliable accurate prenatal non-invasive diagnosis (RAPID) protocol

BackgroundNon-invasive prenatal testing (NIPT) for aneuploidies is now available through commercial companies in many countries, including through private practice in the United Kingdom (UK). Thorough evaluation of service delivery requirements are needed to facilitate NIPT being offered more widely within state funded healthcare systems such as the UK’s National Health Service (NHS). Successful implementation will require the development of laboratory standards, consideration of stakeholder views, an analysis of costs and development of patient and health professional educational materials.Methods/DesignNIPT will be offered in an NHS setting as a contingent screening test. Pregnant woman will be recruited through six maternity units in England and Scotland. Women eligible for Down’s syndrome screening (DSS) will be informed about the study at the time of booking. Women that choose routine DSS will be offered NIPT if they have a screening risk ≥1:1000. NIPT results for trisomy 21, 18, 13 will be reported within 7–10 working days. Data on DSS, NIPT and invasive testing uptake, pregnancy outcomes and test efficacy will be collected. Additional data will be gathered though questionnaires to a) determine acceptability to patients and health professionals, b) evaluate patient and health professional education, c) assess informed choice in women accepting or declining testing and d) gauge family expenses. Qualitative interviews will also be conducted with a sub-set of participating women and health professionals.DiscussionThe results of this study will make a significant contribution to policy decisions around the implementation of NIPT for aneuploidies within the UK NHS. The laboratory standards for testing and reporting, education materials and counselling strategies developed as part of the study are likely to underpin the introduction of NIPT into NHS practice.NIHR Portfolio Number13865

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

RAPIDR: an analysis package for non-invasive prenatal testing of aneuploidy

Non-invasive prenatal testing (NIPT) of fetal aneuploidy using cell-free fetal DNA is becoming part of routine clinical practice. RAPIDR (Reliable Accurate Prenatal non-Invasive Diagnosis R package) is an easy-to-use open-source R package that implements several published NIPT analysis methods. The input to RAPIDR is a set of sequence alignment files in the BAM format, and the outputs are calls for aneuploidy, including trisomies 13, 18, 21 and monosomy X as well as fetal sex. RAPIDR has been extensively tested with a large sample set as part of the RAPID project in the UK. The package contains quality control steps to make it robust for use in the clinical setting. Availability and implementation: RAPIDR is implemented in R and can be freely downloaded via CRAN from here: http://cran.r-project.org/web/packages/RAPIDR/index.html. Contact: kitty.lo@ucl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

A novel homozygous ERCC5 truncating mutation in a family with prenatal arthrogryposis-Further evidence of genotype-phenotype correlation

We report on a family with five fetuses conceived to first cousin parents presenting with abnormal ultrasound findings including contractures and microcephaly. Cerebellar hypoplasia and ventriculomegaly were also present in two and fetal edema developed in the one fetus that survived beyond 24 weeks of gestation. Linkage studies of 15 members of the family, including four affecteds, were undertaken followed by exome sequencing of one affected individual and their parents. Analysis of exome data was restricted to the 9.3 Mb largest shared region of homozygosity identified by linkage; a single novel homozygous mutation in the proband that was heterozygous in the parents (ERCC5 c.2766dupA, p.Leu923ThrfsX7) was identified. This segregated with disease. ERCC5 is a component of the nucleotide excision repair machinery and biallelic mutations in the gene have previously been associated with xeroderma pigmentosum (group G), Cockayne syndrome and the more severe cerebrooculofacioskeletal syndrome. The phenotype in the family we report on is consistent with a severe manifestation of cerebrooculofacioskeletal syndrome. Our data broaden the reported clinical spectrum of ERCC5 mutations and provide further evidence of genotype–phenotype correlation with truncating mutations being associated with severe phenotypes. They also demonstrate the molecular diagnostic power of a combined approach of linkage studies and exome sequencing in families with rare, genetically heterogeneous disorders and a well described pedigree.

Would raising the total cholesterol diagnostic cut-off from 7.5 mmol/L to 9.3 mmol/L improve detection rate of patients with monogenic familial hypercholesterolaemia?

A previous report suggested that 88% of individuals in the general population with total cholesterol (TC) > 9.3 mmol/L have familial hypercholesterolaemia (FH). We tested this hypothesis in a cohort of 4896 UK civil servants, mean (SD) age 44 (±6) years, using next generation sequencing to achieve a comprehensive genetic diagnosis. 25 (0.5%) participants (mean age 49.2 years) had baseline TC > 9.3 mmol/L, and overall we found an FH-causing mutation in the LDLR gene in seven (28%) subjects. The detection rate increased to 39% by excluding eight participants with triglyceride levels over 2.3 mmol/L, and reached 75% in those with TC > 10.4 mmol/L. By extrapolation, the detection rate would be ∼25% by including all participants with TC > 8.6 mmol/L (2.5 standard deviations from the mean). Based on the 1/500 FH frequency, 30% of all FH-cases in this cohort would be missed using the 9.3 mmol/L cut-off. Given that an overall detection rate of 25% is considered economically acceptable, these data suggest that a diagnostic TC cut-off of 8.6 mmol/L, rather than 9.3 mmol/L would be clinically useful for FH in the general population.

Implementing Non-Invasive Prenatal Diagnosis (NIPD) in a National Health Service Laboratory; From Dominant to Recessive Disorders.

Our UK National Health Service regional genetics laboratory offers NIPD for autosomal dominant and de novo conditions (achondroplasia, thanataphoric dysplasia, Apert syndrome), paternal mutation exclusion for cystic fibrosis and a range of bespoke tests. NIPD avoids the risks associated with invasive testing, making prenatal diagnosis more accessible to families at high genetic risk. However, the challenge remains in offering definitive diagnosis for autosomal recessive diseases, which is complicated by the predominance of the maternal mutant allele in the cell-free DNA sample and thus requires a variety of different approaches. Validation and diagnostic implementation for NIPD of congenital adrenal hyperplasia (CAH) is further complicated by presence of a pseudogene that requires a different approach. We have used an assay targeting approximately 6700 heterozygous SNPs around the CAH gene (CYP21A2) to construct the high-risk parental haplotypes and tested this approach in five cases, showing that inheritance of the parental alleles can be correctly identified using NIPD. We are evaluating various measures of the fetal fraction to help determine inheritance of parental mutations. We are currently exploring the utility of an NIPD multi-disorder panel for autosomal recessive disease, to make testing more widely applicable to families with a variety of serious genetic conditions.

Screening for familial hypercholesterolaemia in childhood: Avon Longitudinal Study of Parents and Children (ALSPAC)

Background and aims Familial hypercholesterolaemia (FH) is an autosomal-dominant disease with frequency of 1/500 to 1/250 that leads to premature coronary heart disease. New approaches to identify FH mutation-carriers early are needed to prevent premature cardiac deaths. In a cross-sectional study of the Avon Longitudinal Study of Parents and Children (ALSPAC), we evaluated the biochemical thresholds for FH screening in childhood, and modelled a two-stage biochemical and sequencing screening strategy for FH detection. Methods From 5083 ALSPAC children with cholesterol measurement at age nine years, FH genetic diagnosis was performed in 1512 individuals, using whole-genome or targeted sequencing of known FH-causing genes. Detection rate (DR) and false-positive rate (FPR) for proposed screening thresholds (total-cholesterol > 1.53, or LDL-C > 1.84 multiples of the median (MoM)) were assessed. Results Six of 1512 sequenced individuals had an FH-causing mutation of whom five had LDL-C > 1.84 MoM, giving a verification-bias corrected DR of 62.5% (95% CI: 25–92), with a FPR of 0.2% (95% CI: 0.1–0.4). The DR for the TC cut-point of 1.53 MoM was 25% (95% CI: 3.2–65.1) with a FPR of 0.4% (95% CI: 0.2–0.6). We estimated 13 of an expected 20 FH mutation carriers (and 13 of the 20 parental carriers) could be detected for every 10,000 children screened, with false-positives reliably excluded by addition of a next generation sequencing step in biochemical screen-positive samples. Conclusions Proposed cholesterol thresholds for childhood FH screening were less accurate than previously estimated. A sequential strategy of biochemical screening followed by targeted sequencing of FH genes in screen-positive children may help mitigate the higher than previously estimated FPR and reduce wasted screening of unaffected parents.

Advantages and pitfalls of an extended gene panel for investigating complex neurometabolic phenotypes

Targeted gene panels can be used to establish molecular diagnoses in paediatric cohorts. Reid et al. report that this approach is accurate, efficient and can be preferred to whole-exome or genome sequencing for patients with neurological symptomatology and clues suggestive of an inherited metabolic disorder.