Publications by authors named "Diana Baralle"

74 Publications

Altered regulation of BRCA1 exon 11 splicing is associated with breast cancer risk in carriers of BRCA1 pathogenic variants.

Hum Mutat 2021 Aug 22. Epub 2021 Aug 22.

Department of Public Health and Primary Care, Centre for Cancer Genetic Epidemiology, University of Cambridge, Cambridge, UK.

Germline pathogenic variants in BRCA1 confer a high risk of developing breast and ovarian cancer. The BRCA1 exon 11 (formally exon 10) is one of the largest exons and codes for the nuclear localization signals of the corresponding gene product. This exon can be partially or entirely skipped during pre-mRNA splicing, leading to three major in-frame isoforms that are detectable in most cell types and tissue, and in normal and cancer settings. However, it is unclear whether the splicing imbalance of this exon is associated with cancer risk. Here we identify a common genetic variant in intron 10, rs5820483 (NC_000017.11:g.43095106_43095108dup), which is associated with exon 11 isoform expression and alternative splicing, and with the risk of breast cancer, but not ovarian cancer, in BRCA1 pathogenic variant carriers. The identification of this genetic effect was confirmed by analogous observations in mouse cells and tissue in which a loxP sequence was inserted in the syntenic intronic region. The prediction that the rs5820483 minor allele variant would create a binding site for the splicing silencer hnRNP A1 was confirmed by pull-down assays. Our data suggest that perturbation of BRCA1 exon 11 splicing modifies the breast cancer risk conferred by pathogenic variants of this gene.
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http://dx.doi.org/10.1002/humu.24276DOI Listing
August 2021

Splicing in the Diagnosis of Rare Disease: Advances and Challenges.

Front Genet 2021 1;12:689892. Epub 2021 Jul 1.

School of Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.

Mutations which affect splicing are significant contributors to rare disease, but are frequently overlooked by diagnostic sequencing pipelines. Greater ascertainment of pathogenic splicing variants will increase diagnostic yields, ending the diagnostic odyssey for patients and families affected by rare disorders, and improving treatment and care strategies. Advances in sequencing technologies, predictive modeling, and understanding of the mechanisms of splicing in recent years pave the way for improved detection and interpretation of splice affecting variants, yet several limitations still prohibit their routine ascertainment in diagnostic testing. This review explores some of these advances in the context of clinical application and discusses challenges to be overcome before these variants are comprehensively and routinely recognized in diagnostics.
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http://dx.doi.org/10.3389/fgene.2021.689892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8280750PMC
July 2021

Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly.

Genome Med 2021 02 25;13(1):34. Epub 2021 Feb 25.

Wessex Clinical Genetics Service, Princess Anne Hospital, University Hospital Southampton NHS Foundation Trust, Coxford Rd, Southampton, SO165YA, UK.

Background: Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as "coatopathies".

Methods: Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1's splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2's missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2's mutation.

Results: We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between β-COP and β'-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant β-COP, with the mutant protein being retarded in the Golgi.

Conclusions: This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.
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http://dx.doi.org/10.1186/s13073-021-00850-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908744PMC
February 2021

A novel ACE2 isoform is expressed in human respiratory epithelia and is upregulated in response to interferons and RNA respiratory virus infection.

Nat Genet 2021 02 11;53(2):205-214. Epub 2021 Jan 11.

Faculty of Medicine, School of Clinical and Experimental Sciences, University of Southampton, Southampton, UK.

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in airway epithelial cells for SARS-CoV-2. ACE2 binding to the SARS-CoV-2 protein spike triggers viral fusion with the cell plasma membrane, resulting in viral RNA genome delivery into the host. Despite ACE2's critical role in SARS-CoV-2 infection, full understanding of ACE2 expression, including in response to viral infection, remains unclear. ACE2 was thought to encode five transcripts and one protein of 805 amino acids. In the present study, we identify a novel short isoform of ACE2 expressed in the airway epithelium, the main site of SARS-CoV-2 infection. Short ACE2 is substantially upregulated in response to interferon stimulation and rhinovirus infection, but not SARS-CoV-2 infection. This short isoform lacks SARS-CoV-2 spike high-affinity binding sites and, altogether, our data are consistent with a model where short ACE2 is unlikely to directly contribute to host susceptibility to SARS-CoV-2 infection.
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http://dx.doi.org/10.1038/s41588-020-00759-xDOI Listing
February 2021

Pathogenic variants causing ABL1 malformation syndrome cluster in a myristoyl-binding pocket and increase tyrosine kinase activity.

Eur J Hum Genet 2021 Apr 22;29(4):593-603. Epub 2020 Nov 22.

Wessex Clinical Genetics Service, University Hospital Southampton NHS Foundation Trust, Southampton, UK.

ABL1 is a proto-oncogene encoding a nonreceptor tyrosine kinase, best known in the somatic BCR-ABL fusion gene associated with chronic myeloid leukaemia. Recently, germline missense variants in ABL1 have been found to cause an autosomal dominant developmental syndrome with congenital heart disease, skeletal malformations and characteristic facies. Here, we describe a series of six new unrelated individuals with heterozygous missense variants in ABL1 (including four novel variants) identified via whole exome sequencing. All the affected individuals in this series recapitulate the phenotype of the ABL1 developmental syndrome and additionally we affirm that hearing impairment is a common feature of the condition. Four of the variants cluster in the myristoyl-binding pocket of ABL1, a region critical for auto-inhibitory regulation of the kinase domain. Bio-informatic analysis of transcript-wide conservation and germline/somatic variation reveals that this pocket region is subject to high missense constraint and evolutionary conservation. Functional work to investigate ABL1 kinase activity in vitro by transient transfection of HEK293T cells with variant ABL1 plasmid constructs revealed increased phosphorylation of ABL1-specific substrates compared to wild-type. The increased tyrosine kinase activity was suppressed by imatinib treatment. This case series of six new patients with germline heterozygous ABL1 missense variants further delineates the phenotypic spectrum of this condition and recognises microcephaly as a common finding. Our analysis supports an ABL1 gain-of-function mechanism due to loss of auto-inhibition, and demonstrates the potential for pharmacological inhibition using imatinib.
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http://dx.doi.org/10.1038/s41431-020-00766-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115115PMC
April 2021

'Next Generation Sequencing' as a diagnostic tool in paediatrics.

Arch Dis Child 2021 01 29;106(1):1-2. Epub 2020 Oct 29.

Clinical Genetics, Southampton University Hospitals NHS Trust, Southampton, UK.

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http://dx.doi.org/10.1136/archdischild-2020-320251DOI Listing
January 2021

Correction: Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance.

Genet Med 2020 Jun;22(6):1129

Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41436-020-0789-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272324PMC
June 2020

Novel truncating mutations in CTNND1 cause a dominant craniofacial and cardiac syndrome.

Hum Mol Genet 2020 07;29(11):1900-1921

Centre for Craniofacial and Regenerative Biology, Faculty of Dentistry, Oral and Craniofacial Sciences, King's College London, London SE1 9RT, UK.

CTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, including the maintenance of cell-cell junctions, regulation of the epithelial-mesenchymal transition and transcriptional signalling. Due to advances in next-generation sequencing, CTNND1 has been implicated in human diseases including cleft palate and blepharocheilodontic (BCD) syndrome albeit only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in 13 participants from nine families presenting with craniofacial dysmorphisms including cleft palate and hypodontia, as well as congenital cardiac anomalies, limb dysmorphologies and neurodevelopmental disorders. Using conditional deletions in mice as well as CRISPR/Cas9 approaches to target CTNND1 in Xenopus, we identified a subset of phenotypes that can be linked to p120-catenin in epithelial integrity and turnover, and additional phenotypes that suggest mesenchymal roles of CTNND1. We propose that CTNND1 variants have a wider developmental role than previously described and that variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader velocardiofacial-like syndrome.
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http://dx.doi.org/10.1093/hmg/ddaa050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372553PMC
July 2020

Essentiality-specific pathogenicity prioritization gene score to improve filtering of disease sequence data.

Brief Bioinform 2021 03;22(2):1782-1789

The causal genetic variants underlying more than 50% of single gene (monogenic) disorders are yet to be discovered. Many patients with conditions likely to have a monogenic basis do not receive a confirmed molecular diagnosis which has potential impacts on clinical management. We have developed a gene-specific score, essentiality-specific pathogenicity prioritization (ESPP), to guide the recognition of genes likely to underlie monogenic disease variation to assist in filtering of genome sequence data. When a patient genome is sequenced, there are frequently several plausibly pathogenic variants identified in different genes. Recognition of the single gene most likely to include pathogenic variation can guide the identification of a causal variant. The ESPP score integrates gene-level scores which are broadly related to gene essentiality. Previous work towards the recognition of monogenic disease genes proposed a model with increasing gene essentiality from 'non-essential' to 'essential' genes (for which pathogenic variation may be incompatible with survival) with genes liable to contain disease variation positioned between these two extremes. We demonstrate that the ESPP score is useful for recognizing genes with high potential for pathogenic disease-related variation. Genes classed as essential have particularly high scores, as do genes recently recognized as strong candidates for developmental disorders. Through the integration of individual gene-specific scores, which have different properties and assumptions, we demonstrate the utility of an essentiality-based gene score to improve sequence genome filtering.
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http://dx.doi.org/10.1093/bib/bbaa029DOI Listing
March 2021

Cancer Variant Interpretation Group UK (CanVIG-UK): an exemplar national subspecialty multidisciplinary network.

J Med Genet 2020 12 13;57(12):829-834. Epub 2020 Mar 13.

Division of Genetics and Epidemiology, Institute of Cancer Research, Sutton, UK

Advances in technology have led to a massive expansion in the capacity for genomic analysis, with a commensurate fall in costs. The clinical indications for genomic testing have evolved markedly; the volume of clinical sequencing has increased dramatically; and the range of clinical professionals involved in the process has broadened. There is general acceptance that our early dichotomous paradigms of variants being pathogenic-high risk and benign-no risk are overly simplistic. There is increasing recognition that the clinical interpretation of genomic data requires significant expertise in disease-gene-variant associations specific to each disease area. Inaccurate interpretation can lead to clinical mismanagement, inconsistent information within families and misdirection of resources. It is for this reason that 'national subspecialist multidisciplinary meetings' (MDMs) for genomic interpretation have been articulated as key for the new NHS Genomic Medicine Service, of which Cancer Variant Interpretation Group UK (CanVIG-UK) is an early exemplar. CanVIG-UK was established in 2017 and now has >100 UK members, including at least one clinical diagnostic scientist and one clinical cancer geneticist from each of the 25 regional molecular genetics laboratories of the UK and Ireland. Through CanVIG-UK, we have established national consensus around variant interpretation for cancer susceptibility genes via monthly national teleconferenced MDMs and collaborative data sharing using a secure online portal. We describe here the activities of CanVIG-UK, including exemplar outputs and feedback from the membership.
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http://dx.doi.org/10.1136/jmedgenet-2019-106759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691806PMC
December 2020

Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance.

Genet Med 2020 06 3;22(6):1005-1014. Epub 2020 Mar 3.

Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK.

Purpose: Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories.

Methods: Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software.

Results: Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity.

Conclusion: Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.
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http://dx.doi.org/10.1038/s41436-020-0766-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272326PMC
June 2020

Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders.

Am J Hum Genet 2020 03 27;106(3):338-355. Epub 2020 Feb 27.

Developmental Brain Disorders Laboratory, INSERM UMR 1163, 75015 Paris, France; Service de Génétique, Necker Enfants Malades University Hospital, Assistance Publique-Hôpitaux de Pairs, 75015 Paris, France.

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper- or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.
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http://dx.doi.org/10.1016/j.ajhg.2020.01.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058823PMC
March 2020

Mutation spectrum of PRPF31, genotype-phenotype correlation in retinitis pigmentosa, and opportunities for therapy.

Exp Eye Res 2020 03 31;192:107950. Epub 2020 Jan 31.

Department of Engineering, Design and Mathematics, University of the West of England, Bristol, UK.

Pathogenic variants in pre-messenger RNA (pre-mRNA) splicing factor 31, PRPF31, are the second most common genetic cause of autosomal dominant retinitis pigmentosa (adRP) in most populations. This remains a completely untreatable and incurable form of blindness, and it can be difficult to predict the clinical course of disease. In order to design appropriate targeted therapies, a thorough understanding of the genetics and molecular mechanism of this disease is required. Here, we present the structure of the PRPF31 gene and PRPF31 protein, current understanding of PRPF31 protein function and the full spectrum of all reported clinically relevant variants in PRPF31. We delineate the correlation between specific PRPF31 genotype and RP phenotype, suggesting that, except in cases of complete gene deletion or large-scale deletions, dominant negative effects contribute to phenotype as well as haploinsufficiency. This has important impacts on design of targeted therapies, particularly the feasibility of gene augmentation as a broad approach for treatment of PRPF31-associated RP. We discuss other opportunities for therapy, including antisense oligonucleotide therapy and gene-independent approaches and offer future perspectives on treatment of this form of RP.
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http://dx.doi.org/10.1016/j.exer.2020.107950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065041PMC
March 2020

Exploring the RNA Gap for Improving Diagnostic Yield in Primary Immunodeficiencies.

Front Genet 2019 11;10:1204. Epub 2019 Dec 11.

University of Southampton Medical School, University of Southampton, Southampton, United Kingdom.

Challenges in diagnosing primary immunodeficiency are numerous and diverse, with current whole-exome and whole-genome sequencing approaches only able to reach a molecular diagnosis in 25-60% of cases. We assess these problems and discuss how RNA-focused analysis has expanded and improved in recent years and may now be utilized to gain an unparalleled insight into cellular immunology. We review how investigation into RNA biology can give information regarding the differential expression, monoallelic expression, and alternative splicing-which have important roles in immune regulation and function. We show how this information can inform bioinformatic analysis pipelines and aid in the variant filtering process, expediting the identification of causal variants-especially those affecting splicing-and enhance overall diagnostic ability. We also demonstrate the challenges, which remain in the design of this type of investigation, regarding technological limitation and biological considerations and suggest potential directions for the clinical applications.
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http://dx.doi.org/10.3389/fgene.2019.01204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917654PMC
December 2019

Machine Learning Approaches for the Prioritization of Genomic Variants Impacting Pre-mRNA Splicing.

Cells 2019 11 26;8(12). Epub 2019 Nov 26.

North West Genomic Laboratory Hub, Manchester Centre for Genomic Medicine, Manchester University Hospitals NHS Foundation Trust, St Mary's Hospital, Manchester M13 9WJ, UK.

Defects in pre-mRNA splicing are frequently a cause of Mendelian disease. Despite the advent of next-generation sequencing, allowing a deeper insight into a patient's variant landscape, the ability to characterize variants causing splicing defects has not progressed with the same speed. To address this, recent years have seen a sharp spike in the number of splice prediction tools leveraging machine learning approaches, leaving clinical geneticists with a plethora of choices for in silico analysis. In this review, some basic principles of machine learning are introduced in the context of genomics and splicing analysis. A critical comparative approach is then used to describe seven recent machine learning-based splice prediction tools, revealing highly diverse approaches and common caveats. We find that, although great progress has been made in producing specific and sensitive tools, there is still much scope for personalized approaches to prediction of variant impact on splicing. Such approaches may increase diagnostic yields and underpin improvements to patient care.
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http://dx.doi.org/10.3390/cells8121513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953098PMC
November 2019

Null variants and deletions in BRWD3 cause an X-linked syndrome of mild-moderate intellectual disability, macrocephaly, and obesity: A series of 17 patients.

Am J Med Genet C Semin Med Genet 2019 12 12;181(4):638-643. Epub 2019 Nov 12.

South West Thames Regional Genetics Service, St George's University NHS Foundation Trust, London, UK.

BRWD3 has been described as a cause of X-linked intellectual disability, but relatively little is known about the specific phenotype. We report the largest BRWD3 patient series to date, comprising 17 males with 12 distinct null variants and 2 partial gene deletions. All patients presented with intellectual disability, which was classified as moderate (65%) or mild (35%). Behavioral issues were present in 75% of patients, including aggressive behavior, attention deficit/hyperactivity and/or autistic spectrum disorders. Mean head circumference was +2.8 SD (2.8 standard deviations above the mean), and mean BMI was +2.0 SD (in the context of a mean height of +1.3 SD), indicating a predominant macrocephaly/obesity phenotype. Shared facial features included a tall chin, prognathism, broad forehead, and prominent supraorbital ridge. Additional features, reported in a minority (<30%) of patients included cryptorchidism, neonatal hypotonia, and small joint hypermobility. This study delineates the clinical features associated with BRWD3 null variants and partial gene deletions, and suggests that BRWD3 should be included in the differential diagnosis of patients with an overgrowth-intellectual disability (OGID) phenotype, particularly in male patients with a mild or moderate intellectual disability associated with macrocephaly and/or obesity.
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http://dx.doi.org/10.1002/ajmg.c.31750DOI Listing
December 2019

Splicing in the pathogenesis, diagnosis and treatment of ciliopathies.

Biochim Biophys Acta Gene Regul Mech 2019 Nov - Dec;1862(11-12):194433. Epub 2019 Nov 4.

Faculty of Medicine, University of Southampton, Human Development and Health, United Kingdom of Great Britain and Northern Ireland; University Hospital Southampton NHS Foundation Trust, United Kingdom of Great Britain and Northern Ireland. Electronic address:

Primary cilia are essential signalling organelles found on the apical surface of epithelial cells, where they coordinate chemosensation, mechanosensation and light sensation. Motile cilia play a central role in establishing fluid flow in the respiratory tract, reproductive tract, brain ventricles and ear. Genetic defects affecting the structure or function of cilia can lead to a broad range of developmental and degenerative diseases known as ciliopathies. Splicing contributes to the pathogenesis, diagnosis and treatment of ciliopathies. Tissue-specific alternative splicing contributes to the tissue-specific manifestation of ciliopathy phenotypes, for example the retinal-specific effects of some genetic defects, due to specific transcript expression in the highly specialised ciliated cells of the retina, the photoreceptor cells. Ciliopathies can arise both as a result of genetic variants in spliceosomal proteins, or as a result of variants affecting splicing of specific cilia genes. Here we discuss the opportunities and challenges in diagnosing ciliopathies using RNA sequence analysis and the potential for treating ciliopathies in a relatively mutation-neutral way by targeting splicing. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.
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http://dx.doi.org/10.1016/j.bbagrm.2019.194433DOI Listing
February 2020

A small gene sequencing panel realises a high diagnostic rate in patients with congenital nystagmus following basic phenotyping.

Sci Rep 2019 09 13;9(1):13229. Epub 2019 Sep 13.

Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, MP806, Tremona Road, Southampton, SO16 6YD, UK.

Nystagmus is a disorder of uncontrolled eye movement and can occur as an isolated trait (idiopathic INS, IINS) or as part of multisystem disorders such as albinism, significant visual disorders or neurological disease. Eighty-one unrelated patients with nystagmus underwent routine ocular phenotyping using commonly available phenotyping methods and were grouped into four sub-cohorts according to the level of phenotyping information gained and their findings. DNA was extracted and sequenced using a broad utility next generation sequencing (NGS) gene panel. A clinical subpanel of genes for nystagmus/albinism was utilised and likely causal variants were prioritised according to methods currently employed by clinical diagnostic laboratories. We determine the likely underlying genetic cause for 43.2% of participants with similar yields regardless of prior phenotyping. This study demonstrates that a diagnostic workflow combining basic ocular phenotyping and a clinically available targeted NGS panel, can provide a high diagnostic yield for patients with infantile nystagmus, enabling access to disease specific management at a young age and reducing the need for multiple costly, often invasive tests. By describing diagnostic yield for groups of patients with incomplete phenotyping data, it also permits the subsequent design of 'real-world' diagnostic workflows and illustrates the changing role of genetic testing in modern diagnostic workflows for heterogeneous ophthalmic disorders.
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http://dx.doi.org/10.1038/s41598-019-49368-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744446PMC
September 2019

Exome sequencing in patients with antiepileptic drug exposure and complex phenotypes.

Arch Dis Child 2020 04 3;105(4):384-389. Epub 2019 Sep 3.

Genetic Medicine, Central Manchester University Hospitals Foundation Trust, Manchester, UK.

Introduction: Fetal anticonvulsant syndrome (FACS) describes the pattern of physical and developmental problems seen in those children exposed to certain antiepileptic drugs (AEDs) in utero. The diagnosis of FACS is a clinical one and so excluding alternative diagnoses such as genetic disorders is essential.

Methods: We reviewed the pathogenicity of reported variants identified on exome sequencing in the Deciphering Developmental Disorders (DDD) Study in 42 children exposed to AEDs in utero, but where a diagnosis other than FACS was suspected. In addition, we analysed chromosome microarray data from 10 patients with FACS seen in a Regional Genetics Service.

Results: Seven children (17%) from the DDD Study had a copy number variant or pathogenic variant in a developmental disorder gene which was considered to explain or partially explain their phenotype. Across the AED exposure types, variants were found in 2/15 (13%) valproate exposed cases and 3/14 (21%) carbamazepine exposed cases. No pathogenic copy number variants were identified in our local sample (n=10).

Conclusions: This study is the first of its kind to analyse the exomes of children with developmental disorders who were exposed to AEDs in utero. Though we acknowledge that the results are subject to bias, a significant number of children were identified with alternate diagnoses which had an impact on counselling and management. We suggest that consideration is given to performing whole exome sequencing as part of the diagnostic work-up for children exposed to AEDs in utero.
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http://dx.doi.org/10.1136/archdischild-2018-316547DOI Listing
April 2020

Deleterious de novo variants of X-linked ZC4H2 in females cause a variable phenotype with neurogenic arthrogryposis multiplex congenita.

Hum Mutat 2019 12 21;40(12):2270-2285. Epub 2019 Aug 21.

Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway.

Pathogenic variants in the X-linked gene ZC4H2, which encodes a zinc-finger protein, cause an infrequently described syndromic form of arthrogryposis multiplex congenita (AMC) with central and peripheral nervous system involvement. We present genetic and detailed phenotypic information on 23 newly identified families and simplex cases that include 19 affected females from 18 families and 14 affected males from nine families. Of note, the 15 females with deleterious de novo ZC4H2 variants presented with phenotypes ranging from mild to severe, and their clinical features overlapped with those seen in affected males. By contrast, of the nine carrier females with inherited ZC4H2 missense variants that were deleterious in affected male relatives, four were symptomatic. We also compared clinical phenotypes with previously published cases of both sexes and provide an overview on 48 males and 57 females from 42 families. The spectrum of ZC4H2 defects comprises novel and recurrent mostly inherited missense variants in affected males, and de novo splicing, frameshift, nonsense, and partial ZC4H2 deletions in affected females. Pathogenicity of two newly identified missense variants was further supported by studies in zebrafish. We propose ZC4H2 as a good candidate for early genetic testing of males and females with a clinical suspicion of fetal hypo-/akinesia and/or (neurogenic) AMC.
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http://dx.doi.org/10.1002/humu.23841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874899PMC
December 2019

Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging.

Genome Res 2019 07 3;29(7):1057-1066. Epub 2019 Jun 3.

Institute of Biomedical and Clinical Science, University of Exeter Medical School, RILD Wellcome Wolfson Centre, Royal Devon and Exeter NHS Foundation Trust, Exeter, EX2 5DW, United Kingdom.

Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha () are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: Sotos syndrome overgrowth disorder and Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders.
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http://dx.doi.org/10.1101/gr.243584.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633263PMC
July 2019

Phenotypic and biochemical analysis of an international cohort of individuals with variants in NAA10 and NAA15.

Hum Mol Genet 2019 09;28(17):2900-2919

Department of Human Genetics, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA.

N-alpha-acetylation is one of the most common co-translational protein modifications in humans and is essential for normal cell function. NAA10 encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. The auxiliary and regulatory subunits of the NatA complex are NAA15 and Huntington-interacting protein (HYPK), respectively. Through a genotype-first approach with exome sequencing, we identified and phenotypically characterized 30 individuals from 30 unrelated families with 17 different de novo or inherited, dominantly acting missense variants in NAA10 or NAA15. Clinical features of affected individuals include variable levels of intellectual disability, delayed speech and motor milestones and autism spectrum disorder. Additionally, some subjects present with mild craniofacial dysmorphology, congenital cardiac anomalies and seizures. One of the individuals is an 11-year-old boy with a frameshift variant in exon 7 of NAA10, who presents most notably with microphthalmia, which confirms a prior finding with a single family with Lenz microphthalmia syndrome. Biochemical analyses of variants as part of the human NatA complex, as well as enzymatic analyses with and without the HYPK regulatory subunit, help to explain some of the phenotypic differences seen among the different variants.
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http://dx.doi.org/10.1093/hmg/ddz111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736318PMC
September 2019

A Syndromic Neurodevelopmental Disorder Caused by Mutations in SMARCD1, a Core SWI/SNF Subunit Needed for Context-Dependent Neuronal Gene Regulation in Flies.

Am J Hum Genet 2019 04 14;104(4):596-610. Epub 2019 Mar 14.

Centre Hospitalier Universitaire Sainte-Justine Research Center, University of Montreal, Montreal, QC H3T 1C5, Canada; Department of Pediatrics, University of Montreal, Montreal, QC H4A 3J1, Canada. Electronic address:

Mutations in several genes encoding components of the SWI/SNF chromatin remodeling complex cause neurodevelopmental disorders (NDDs). Here, we report on five individuals with mutations in SMARCD1; the individuals present with developmental delay, intellectual disability, hypotonia, feeding difficulties, and small hands and feet. Trio exome sequencing proved the mutations to be de novo in four of the five individuals. Mutations in other SWI/SNF components cause Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, or other syndromic and non-syndromic NDDs. Although the individuals presented here have dysmorphisms and some clinical overlap with these syndromes, they lack their typical facial dysmorphisms. To gain insight into the function of SMARCD1 in neurons, we investigated the Drosophila ortholog Bap60 in postmitotic memory-forming neurons of the adult Drosophila mushroom body (MB). Targeted knockdown of Bap60 in the MB of adult flies causes defects in long-term memory. Mushroom-body-specific transcriptome analysis revealed that Bap60 is required for context-dependent expression of genes involved in neuron function and development in juvenile flies when synaptic connections are actively being formed in response to experience. Taken together, we identify an NDD caused by SMARCD1 mutations and establish a role for the SMARCD1 ortholog Bap60 in the regulation of neurodevelopmental genes during a critical time window of juvenile adult brain development when neuronal circuits that are required for learning and memory are formed.
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http://dx.doi.org/10.1016/j.ajhg.2019.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451697PMC
April 2019

Identification and functional analysis of a novel oculocerebrorenal syndrome of Lowe () gene variant in two pedigrees with varying phenotypes including isolated congenital cataract.

Mol Vis 2018 31;24:847-852. Epub 2018 Dec 31.

Clinical and Experimental Sciences, University of Southampton, UK.

Purpose: To identify the genetic variation in two unrelated probands with congenital cataract and to perform functional analysis of the detected variants.

Methods: Clinical examination and phenotyping, segregation, and functional analysis were performed for the two studied pedigrees.

Results: A novel gene variant (c.1964A>T, p. (Asp655Val)) was identified. This variant causes defects in OCRL protein folding and mislocalization to the cytoplasm. In addition, the variant's location close to the Rab binding site is likely to be associated with membrane targeting abnormalities.

Conclusions: The results highlight the importance of early genetic diagnosis in infants with congenital cataract and show that mutations in the gene can present as apparently isolated congenital cataract.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334980PMC
May 2019

RNA splicing analysis in genomic medicine.

Int J Biochem Cell Biol 2019 03 27;108:61-71. Epub 2018 Dec 27.

Human Development and Health, Faculty of Medicine, University of Southampton, UK; Wessex Clinical Genetics Service, University Hospital Southampton NHS Foundation Trust, Southampton, UK. Electronic address:

High-throughput next-generation sequencing technologies have led to a rapid increase in the number of sequence variants identified in clinical practice via diagnostic genetic tests. Current bioinformatic analysis pipelines fail to take adequate account of the possible splicing effects of such variants, particularly where variants fall outwith canonical splice site sequences, and consequently the pathogenicity of such variants may often be missed. The regulation of splicing is highly complex and as a result, in silico prediction tools lack sufficient sensitivity and specificity for reliable use. Variants of all kinds can be linked to aberrant splicing in disease and the need for correct identification and diagnosis grows ever more crucial as novel splice-switching antisense oligonucleotide therapies start to enter clinical usage. RT-PCR provides a useful targeted assay of the splicing effects of identified variants, while minigene assays, massive parallel reporter assays and animal models can also be used for more detailed study of a particular splicing system, given enough time and resources. However, RNA-sequencing (RNA-seq) has the potential to be used as a rapid diagnostic tool in genomic medicine. By utilising data science approaches and machine learning, it may prove possible to finally understand and interpret the 'splicing code' and apply this knowledge in human disease diagnostics.
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http://dx.doi.org/10.1016/j.biocel.2018.12.009DOI Listing
March 2019

Breast cancer risk in neurofibromatosis type 1 is a function of the type of gene mutation: a new genotype-phenotype correlation.

J Med Genet 2019 04 10;56(4):209-219. Epub 2018 Dec 10.

Inherited Tumour Syndromes Research Group, Institute of Cancer & Genetics, Cardiff University, Cardiff, UK.

Background: Neurofibromatosis type 1 (NF1) predisposes to breast cancer (BC), but no genotype-phenotype correlations have been described.

Methods: Constitutional mutations in 78 patients with NF1 with BC (NF1-BC) were compared with the Leiden Open Variation Database (n=3432).

Results: No cases were observed with whole or partial gene deletions (HR 0.10; 95% CI 0.006 to 1.63; p=0.014, Fisher's exact test). There were no gross relationships with mutation position. Forty-five (64.3%; HR 6.4-83) of the 70 different mutations were more frequent than expected (p<0.05), while 52 (74.3%; HR 5.3-83) were significant when adjusted for multiple comparisons (adjusted p≤0.125; Benjamini-Hochberg). Higher proportions of both nonsense and missense mutations were also observed (adjusted p=0.254; Benjamini-Hochberg). Ten of the 11 missense cases with known age of BC occurred at <50 years (p=0.041). Eighteen cases had testing, revealing one mutation.

Discussion: These data strongly support the hypothesis that certain constitutional mutation types, and indeed certain specific variants in confer different risks of BC. The lack of large deletions and excess of nonsenses and missenses is consistent with gain of function mutations conferring risk of BC, and also that neurofibromin may function as a dimer. The observation that somatic amplification can occur independently of amplification in sporadic BC supports this concept. A prospective clinical-molecular study of NF1-BC needs to be established to confirm and build on these findings, but regardless of mutation status patients with NF1-BC warrant testing of other BC-predisposing genes.
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http://dx.doi.org/10.1136/jmedgenet-2018-105599DOI Listing
April 2019

Gene-specific metrics to facilitate identification of disease genes for molecular diagnosis in patient genomes: a systematic review.

Brief Funct Genomics 2019 02;18(1):23-29

Genetic Epidemiology and Bioinformatics Research Group, Human Development and Health, Faculty of Medicine, University of Southampton, UK.

The evolution of next-generation sequencing technologies has facilitated the detection of causal genetic variants in diseases previously undiagnosed at a molecular level. However, in genome sequencing studies, the identification of disease genes among a candidate gene list is often difficult because of the large number of apparently damaging (but usually neutral) variants. A number of variant prioritization tools have been developed to help detect disease-causal sites. However, the results may be misleading as many variants scored as damaging by these tools are often tolerated, and there are inconsistencies in prediction results among the different variant-level prediction tools. Recently, studies have indicated that understanding gene properties might improve detection of genes liable to have associated disease variation and that this information improves molecular diagnostics. The purpose of this systematic review is to evaluate how understanding gene-specific properties might improve filtering strategies in clinical sequence data to prioritize potential disease variants. Improved understanding of the 'disease genome', which includes coding, noncoding and regulatory variation, might help resolve difficult cases. This review provides a comprehensive assessment of existing gene-level approaches, the relationships between measures of gene-pathogenicity and how use of these prediction tools can be developed for molecular diagnostics.
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http://dx.doi.org/10.1093/bfgp/ely033DOI Listing
February 2019

The Tatton-Brown-Rahman Syndrome: A clinical study of 55 individuals with constitutive variants.

Wellcome Open Res 2018 23;3:46. Epub 2018 Apr 23.

Département de Génétique and Centre de Référence Déficiences Intellectuelles de Causes Rares, Assistance Publique - Hôpitaux de Paris , Paris, France.

Tatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as the DNMT3A-overgrowth syndrome, is an overgrowth intellectual disability syndrome first described in 2014 with a report of 13 individuals with constitutive heterozygous variants. Here we have undertaken a detailed clinical study of 55 individuals with variants, including the 13 previously reported individuals. An intellectual disability and overgrowth were reported in >80% of individuals with TBRS and were designated major clinical associations. Additional frequent clinical associations (reported in 20-80% individuals) included an evolving facial appearance with low-set, heavy, horizontal eyebrows and prominent upper central incisors; joint hypermobility (74%); obesity (weight ³2SD, 67%); hypotonia (54%); behavioural/psychiatric issues (most frequently autistic spectrum disorder, 51%); kyphoscoliosis (33%) and afebrile seizures (22%). One individual was diagnosed with acute myeloid leukaemia in teenage years. Based upon the results from this study, we present our current management for individuals with TBRS.
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http://dx.doi.org/10.12688/wellcomeopenres.14430.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964628PMC
April 2018

De Novo and Inherited Loss-of-Function Variants in TLK2: Clinical and Genotype-Phenotype Evaluation of a Distinct Neurodevelopmental Disorder.

Am J Hum Genet 2018 06 31;102(6):1195-1203. Epub 2018 May 31.

Division of Clinical Genomics, Ambry Genetics, Aliso Viejo, CA 92656, USA.

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.
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http://dx.doi.org/10.1016/j.ajhg.2018.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992133PMC
June 2018
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