Genomic newborn screening for rare diseases.

Rare diseases are a leading cause of infant mortality and lifelong disability. To improve outcomes, timely diagnosis and effective treatments are needed. Genomic sequencing has transformed the traditional diagnostic process, providing rapid, accurate and cost-effective genetic diagnoses to many. Incorporating genomic sequencing into newborn screening programmes at the population scale holds the promise of substantially expanding the early detection of treatable rare diseases, with stored genomic data potentially benefitting health over a lifetime and supporting further research. As several large-scale newborn genomic screening projects launch internationally, we review the challenges and opportunities presented, particularly the need to generate evidence of benefit and to address the ethical, legal and psychosocial issues that genomic newborn screening raises.

Scaling national and international improvement in virtual gene panel curation via a collaborative approach to discordance resolution.

Clinical validity assessments of gene-disease associations underpin analysis and reporting in diagnostic genomics, and yet wide variability exists in practice, particularly in use of these assessments for virtual gene panel design and maintenance. Harmonization efforts are hampered by the lack of agreed terminology, agreed gene curation standards, and platforms that can be used to identify and resolve discrepancies at scale. We undertook a systematic comparison of the content of 80 virtual gene panels used in two healthcare systems by multiple diagnostic providers in the United Kingdom and Australia. The process was enabled by a shared curation platform, PanelApp, and resulted in the identification and review of 2,144 discordant gene ratings, demonstrating the utility of sharing structured gene-disease validity assessments and collaborative discordance resolution in establishing national and international consensus.

Finding Diagnostically Useful Patterns in Quantitative Phenotypic Data.

Trio-based whole-exome sequence (WES) data have established confident genetic diagnoses in ∼40% of previously undiagnosed individuals recruited to the Deciphering Developmental Disorders (DDD) study. Here we aim to use the breadth of phenotypic information recorded in DDD to augment diagnosis and disease variant discovery in probands. Median Euclidean distances (mEuD) were employed as a simple measure of similarity of quantitative phenotypic data within sets of ≥10 individuals with plausibly causative de novo mutations (DNM) in 28 different developmental disorder genes. 13/28 (46.4%) showed significant similarity for growth or developmental milestone metrics, 10/28 (35.7%) showed similarity in HPO term usage, and 12/28 (43%) showed no phenotypic similarity. Pairwise comparisons of individuals with high-impact inherited variants to the 32 individuals with causative DNM in ANKRD11 using only growth z-scores highlighted 5 likely causative inherited variants and two unrecognized DNM resulting in an 18% diagnostic uplift for this gene. Using an independent approach, naive Bayes classification of growth and developmental data produced reasonably discriminative models for the 24 DNM genes with sufficiently complete data. An unsupervised naive Bayes classification of 6,993 probands with WES data and sufficient phenotypic information defined 23 in silico syndromes (ISSs) and was used to test a "phenotype first" approach to the discovery of causative genotypes using WES variants strictly filtered on allele frequency, mutation consequence, and evidence of constraint in humans. This highlighted heterozygous de novo nonsynonymous variants in SPTBN2 as causative in three DDD probands.

MED27, SLC6A7, and MPPE1 Variants in a Complex Neurodevelopmental Disorder with Severe Dystonia.

BACKGROUND: Despite advances in next generation sequencing technologies, the identification of variants of uncertain significance (VUS) can often hinder definitive diagnosis in patients with complex neurodevelopmental disorders. OBJECTIVE: The objective of this study was to identify and characterize the underlying cause of disease in a family with two children with severe developmental delay associated with generalized dystonia and episodic status dystonicus, chorea, epilepsy, and cataracts. METHODS: Candidate genes identified by autozygosity mapping and whole-exome sequencing were characterized using cellular and vertebrate model systems. RESULTS: Homozygous variants were found in three candidate genes: MED27, SLC6A7, and MPPE1. Although the patients had features of MED27-related disorder, the SLC6A7 and MPPE1 variants were functionally investigated. SLC6A7 variant in vitro overexpression caused decreased proline transport as a result of reduced cell-surface expression, and zebrafish knockdown of slc6a7 exhibited developmental delay and fragile motor neuron morphology that could not be rescued by L-proline transporter-G396S RNA. Lastly, patient fibroblasts displayed reduced cell-surface expression of glycophosphatidylinositol-anchored proteins linked to MPPE1 dysfunction. CONCLUSIONS: We report a family harboring a homozygous MED27 variant with additional loss-of-function SLC6A7 and MPPE1 gene variants, which potentially contribute to a blended phenotype caused by multilocus pathogenic variants. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

An online compendium of treatable genetic disorders.

More than 4,000 genes have been associated with recognizable Mendelian/monogenic diseases. When faced with a new diagnosis of a rare genetic disorder, health care providers increasingly turn to internet resources for information to understand the disease and direct care. Unfortunately, it can be challenging to find information concerning treatment for rare diseases as key details are scattered across a number of authoritative websites and numerous journal articles. The website and associated mobile device application described in this article begin to address this challenge by providing a convenient, readily available starting point to find treatment information. The site, Rx-genes.com (https://www.rx-genes.com/), is focused on those conditions where the treatment is directed against the mechanism of the disease and thereby alters the natural history of the disease. The website currently contains 633 disease entries that include references to disease information and treatment guidance, a brief summary of treatments, the inheritance pattern, a disease frequency (if known), nonmolecular confirmatory testing (if available), and a link to experimental treatments. Existing entries are continuously updated, and new entries are added as novel treatments appear in the literature.

Evaluating the performance of a clinical genome sequencing program for diagnosis of rare genetic disease, seen through the lens of craniosynostosis.

PURPOSE: Genome sequencing (GS) for diagnosis of rare genetic disease is being introduced into the clinic, but the complexity of the data poses challenges for developing pipelines with high diagnostic sensitivity. We evaluated the performance of the Genomics England 100,000 Genomes Project (100kGP) panel-based pipelines, using craniosynostosis as a test disease. METHODS: GS data from 114 probands with craniosynostosis and their relatives (314 samples), negative on routine genetic testing, were scrutinized by a specialized research team, and diagnoses compared with those made by 100kGP. RESULTS: Sixteen likely pathogenic/pathogenic variants were identified by 100kGP. Eighteen additional likely pathogenic/pathogenic variants were identified by the research team, indicating that for craniosynostosis, 100kGP panels had a diagnostic sensitivity of only 47%. Measures that could have augmented diagnoses were improved calling of existing panel genes (+18% sensitivity), review of updated panels (+12%), comprehensive analysis of de novo small variants (+29%), and copy-number/structural variants (+9%). Recent NHS England recommendations that partially incorporate these measures should achieve 85% overall sensitivity (+38%). CONCLUSION: GS identified likely pathogenic/pathogenic variants in 29.8% of previously undiagnosed patients with craniosynostosis. This demonstrates the value of research analysis and the importance of continually improving algorithms to maximize the potential of clinical GS.

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease.

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.

A Recurrent De Novo Nonsense Variant in ZSWIM6 Results in Severe Intellectual Disability without Frontonasal or Limb Malformations.

A recurrent de novo missense variant within the C-terminal Sin3-like domain of ZSWIM6 was previously reported to cause acromelic frontonasal dysostosis (AFND), an autosomal-dominant severe frontonasal and limb malformation syndrome, associated with neurocognitive and motor delay, via a proposed gain-of-function effect. We present detailed phenotypic information on seven unrelated individuals with a recurrent de novo nonsense variant (c.2737C>T [p.Arg913Ter]) in the penultimate exon of ZSWIM6 who have severe-profound intellectual disability and additional central and peripheral nervous system symptoms but an absence of frontonasal or limb malformations. We show that the c.2737C>T variant does not trigger nonsense-mediated decay of the ZSWIM6 mRNA in affected individual-derived cells. This finding supports the existence of a truncated ZSWIM6 protein lacking the Sin3-like domain, which could have a dominant-negative effect. This study builds support for a key role for ZSWIM6 in neuronal development and function, in addition to its putative roles in limb and craniofacial development, and provides a striking example of different variants in the same gene leading to distinct phenotypes.

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. FUNDING: UK Department of Health and Social Care and The Wellcome Trust.

The Human Phenotype Ontology in 2017.

Deep phenotyping has been defined as the precise and comprehensive analysis of phenotypic abnormalities in which the individual components of the phenotype are observed and described. The three components of the Human Phenotype Ontology (HPO; www.human-phenotype-ontology.org) project are the phenotype vocabulary, disease-phenotype annotations and the algorithms that operate on these. These components are being used for computational deep phenotyping and precision medicine as well as integration of clinical data into translational research. The HPO is being increasingly adopted as a standard for phenotypic abnormalities by diverse groups such as international rare disease organizations, registries, clinical labs, biomedical resources, and clinical software tools and will thereby contribute toward nascent efforts at global data exchange for identifying disease etiologies. This update article reviews the progress of the HPO project since the debut Nucleic Acids Research database article in 2014, including specific areas of expansion such as common (complex) disease, new algorithms for phenotype driven genomic discovery and diagnostics, integration of cross-species mapping efforts with the Mammalian Phenotype Ontology, an improved quality control pipeline, and the addition of patient-friendly terminology.

Mutations in PIEZO2 cause Gordon syndrome, Marden-Walker syndrome, and distal arthrogryposis type 5.

Gordon syndrome (GS), or distal arthrogryposis type 3, is a rare, autosomal-dominant disorder characterized by cleft palate and congenital contractures of the hands and feet. Exome sequencing of five GS-affected families identified mutations in piezo-type mechanosensitive ion channel component 2 (PIEZO2) in each family. Sanger sequencing revealed PIEZO2 mutations in five of seven additional families studied (for a total of 10/12 [83%] individuals), and nine families had an identical c.8057G>A (p.Arg2686His) mutation. The phenotype of GS overlaps with distal arthrogryposis type 5 (DA5) and Marden-Walker syndrome (MWS). Using molecular inversion probes for targeted sequencing to screen PIEZO2, we found mutations in 24/29 (82%) DA5-affected families and one of two MWS-affected families. The presence of cleft palate was significantly associated with c.8057G>A (Fisher's exact test, adjusted p value < 0.0001). Collectively, although GS, DA5, and MWS have traditionally been considered separate disorders, our findings indicate that they are etiologically related and perhaps represent variable expressivity of the same condition.

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.

GABRB3 mutations: a new and emerging cause of early infantile epileptic encephalopathy.

The gamma-aminobutyric acid type A receptor ß3 gene (GABRB3) encodes the ß3-subunit of the gamma-aminobutyric acid type A (GABAA ) receptor, which mediates inhibitory signalling within the central nervous system. Recently, GABRB3 mutations have been identified in a few patients with infantile spasms and Lennox-Gastaut syndrome. We report the clinical and electrographic features of a novel case of GABRB3-related early-onset epileptic encephalopathy. Our patient presented with neonatal hypotonia and feeding difficulties, then developed pharmacoresistant epileptic encephalopathy, characterized by multiple seizure types from 3 months of age. Electroencephalography demonstrated ictal generalized and interictal multifocal epileptiform abnormalities. Using a SureSelectXT custom multiple gene panel covering 48 early infantile epileptic encephalopathy/developmental delay genes, a novel de novo GABRB3 heterozygous missense mutation, c.860C>T (p.Thr287Ile), was identified and confirmed on Sanger sequencing. GABRB3 is an emerging cause of early-onset epilepsy. Novel genetic technologies, such as whole-exome/genome sequencing and multiple gene panels, will undoubtedly identify further cases, allowing more detailed electroclinical delineation of the GABRB3-related genotypic and phenotypic spectra.

The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data.

The Human Phenotype Ontology (HPO) project, available at http://www.human-phenotype-ontology.org, provides a structured, comprehensive and well-defined set of 10,088 classes (terms) describing human phenotypic abnormalities and 13,326 subclass relations between the HPO classes. In addition we have developed logical definitions for 46% of all HPO classes using terms from ontologies for anatomy, cell types, function, embryology, pathology and other domains. This allows interoperability with several resources, especially those containing phenotype information on model organisms such as mouse and zebrafish. Here we describe the updated HPO database, which provides annotations of 7,278 human hereditary syndromes listed in OMIM, Orphanet and DECIPHER to classes of the HPO. Various meta-attributes such as frequency, references and negations are associated with each annotation. Several large-scale projects worldwide utilize the HPO for describing phenotype information in their datasets. We have therefore generated equivalence mappings to other phenotype vocabularies such as LDDB, Orphanet, MedDRA, UMLS and phenoDB, allowing integration of existing datasets and interoperability with multiple biomedical resources. We have created various ways to access the HPO database content using flat files, a MySQL database, and Web-based tools. All data and documentation on the HPO project can be found online.

De novo heterozygous mutations in SMC3 cause a range of Cornelia de Lange syndrome-overlapping phenotypes.

Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations, and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS-like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published. Here, we report the efforts of an international research and clinical collaboration to provide clinical comparison of 16 patients with CdLS-like features caused by mutations in SMC3. Modeling of the mutation effects on protein structure suggests a dominant-negative effect on the multimeric cohesin complex. When compared with typical CdLS, many SMC3-associated phenotypes are also characterized by postnatal microcephaly but with a less distinctive craniofacial appearance, a milder prenatal growth retardation that worsens in childhood, few congenital heart defects, and an absence of limb deficiencies. While most mutations are unique, two unrelated affected individuals shared the same mutation but presented with different phenotypes. This work confirms that de novo SMC3 mutations account for ∼ 1%-2% of CdLS-like phenotypes.

Haploinsufficiency of SF3B4, a component of the pre-mRNA spliceosomal complex, causes Nager syndrome.

Nager syndrome, first described more than 60 years ago, is the archetype of a class of disorders called the acrofacial dysostoses, which are characterized by craniofacial and limb malformations. Despite intensive efforts, no gene for Nager syndrome has yet been identified. In an international collaboration, FORGE Canada and the National Institutes of Health Centers for Mendelian Genomics used exome sequencing as a discovery tool and found that mutations in SF3B4, a component of the U2 pre-mRNA spliceosomal complex, cause Nager syndrome. After Sanger sequencing of SF3B4 in a validation cohort, 20 of 35 (57%) families affected by Nager syndrome had 1 of 18 different mutations, nearly all of which were frameshifts. These results suggest that most cases of Nager syndrome are caused by haploinsufficiency of SF3B4. Our findings add Nager syndrome to a growing list of disorders caused by mutations in genes that encode major components of the spliceosome and also highlight the synergistic potential of international collaboration when exome sequencing is applied in the search for genes responsible for rare Mendelian phenotypes.

Familial recurrences of FOXG1-related disorder: Evidence for mosaicism.

FOXG1-related disorders are caused by heterozygous mutations in FOXG1 and result in a spectrum of neurodevelopmental phenotypes including postnatal microcephaly, intellectual disability with absent speech, epilepsy, chorea, and corpus callosum abnormalities. The recurrence risk for de novo mutations in FOXG1-related disorders is assumed to be low. Here, we describe three unrelated sets of full siblings with mutations in FOXG1 (c.515_577del63, c.460dupG, and c.572T > G), representing familial recurrence of the disorder. In one family, we have documented maternal somatic mosaicism for the FOXG1 mutation, and all of the families presumably represent parental gonadal (or germline) mosaicism. To our knowledge, mosaicism has not been previously reported in FOXG1-related disorders. Therefore, this report provides evidence that germline mosaicism for FOXG1 mutations is a likely explanation for familial recurrence and should be considered during recurrence risk counseling for families of children with FOXG1-related disorders.

Exome sequencing for prenatal diagnosis of fetuses with sonographic abnormalities.

OBJECTIVE: 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. METHOD: Whole exome sequencing was performed on DNA extracted from chorionic villi or amniocytes in 24 fetuses with unexplained ultrasound findings. In the first 14 cases sequencing was initially performed on fetal DNA only. For the remaining 10, the trio of fetus, mother and father was sequenced simultaneously. RESULTS: In 21% (5/24) cases, exome sequencing provided definitive diagnoses (Milroy disease, hypophosphatasia, achondrogenesis type 2, Freeman-Sheldon syndrome and Baraitser-Winter Syndrome). In a further case, a plausible diagnosis of orofaciodigital syndrome type 6 was made. In two others, a single mutation in an autosomal recessive gene was identified, but incomplete sequencing coverage precluded exclusion of the presence of a second mutation. CONCLUSION: Whole exome sequencing improves prenatal diagnosis in euploid fetuses with abnormal ultrasound scans. In order to expedite interpretation of results, trio sequencing should be employed, but interpretation can still be compromised by incomplete coverage of relevant genes.