Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments

A Systematic Review of Occupational Exposure to Particulate Matter and Cardiovascular Disease

Exposure to ambient particulate air pollution is a recognized risk factor for cardiovascular disease; however the link between occupational particulate exposures and adverse cardiovascular events is less clear. We conducted a systematic review, including meta-analysis where appropriate, of the epidemiologic association between occupational exposure to particulate matter and cardiovascular disease. Out of 697 articles meeting our initial criteria, 37 articles published from January 1990 to April 2009 (12 mortality; 5 morbidity; and 20 intermediate cardiovascular endpoints) were included. Results suggest a possible association between occupational particulate exposures and ischemic heart disease (IHD) mortality as well as non-fatal myocardial infarction (MI), and stronger evidence of associations with heart rate variability and systemic inflammation, potential intermediates between occupational PM exposure and IHD. In meta-analysis of mortality studies, a significant increase in IHD was observed (meta-IRR = 1.16; 95% CI: 1.06–1.26), however these data were limited by lack of adequate control for smoking and other potential confounders. Further research is needed to better clarify the magnitude of the potential risk of the development and aggravation of IHD associated with short and long-term occupational particulate exposures and to clarify the clinical significance of acute and chronic changes in intermediate cardiovascular outcomes

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.</p

Decoding the Genetics of Inherited Retinal Diseases in Ireland

Title: Decoding the Genetics of Inherited Retinal Diseases in Ireland Short Description: The Target 5000 study aims to provide genetic testing for the estimated 5,000 people in Ireland who are affected by a genetic retinal condition. Currently, nearly 1000 patients in Ireland have been sequenced and approximately 80 novel candidate variants have been discovered. Purpose: While clinical trials are in progress for patients with inherited retinal degenerations (IRDs), many such trials require patients to have a known causative mutation to participate in these trials. The Target 5000 research project aims to genetically characterise the estimated 5,000 people in Ireland affected by IRDs. Approach: The IRD patient cohort used in the study has been obtained via a collaborative network of ophthalmologists whereby if an IRD is suspected, given consent, a DNA sample is taken and provided to a central laboratory for genetic analysis. The study seeks to detect previously identified, together with as yet undiscovered, pathological mutations in a panel of known retinal degeneration genes utilizing target capture next generation sequencing (NGS). Where a candidate variant is identified by NGS, additional family members are recruited to verify the appropriate presence and segregation of the variant with the relevant condition. The study to date includes data from nearly 1000 IRD patients. Results: To date, nearly 1000 patients from the Irish IRD population have been sequenced for 254 genes, providing real insights into the genetic architecture of IRDs in Ireland. Target 5000 offers not only a chance to discover new relevant and pathogenic mutations but is vital to providing patients with information regarding the underlying genetic pathogenesis of their disease. Over 80 novel IRD mutations have been discovered to date, this includes several novel large structural variants such as inversions and deletions spanning several kilobases. The findings have also proved useful in the clinical setting where candidate variants have helped to resolve some previously ambiguous disease phenotypes. The study has also led to the identification of several actionable variants in our cohort where there is active recruitment for natural history studies or clinical trials of new therapeutics. Conclusions: The aim of the study is to genetically characterise patients with IRDs in Ireland and in principle potentially enable clinical trials to be more accessible for some patients analysed where appropriate. Thus far, during the study, genetic analysis of IRD patients has helped to resolve ambiguous phenotypes and to identify causative mutations in nearly 70% of cases. The continuous growth of the cohort has enabled us to better analyse the sequencing data and interpret the potential pathogenicity of novel variants when detected. In addition to this, the growing body of data from NGS studies of IRDs globally should facilitate better correlations between genotype and phenotype and further refine methods for diagnoses and prognoses

Next-Generation Sequencing Applications for Inherited Retinal Diseases

Inherited retinal diseases (IRDs) represent a collection of phenotypically and genetically diverse conditions. IRDs phenotype(s) can be isolated to the eye or can involve multiple tissues. These conditions are associated with diverse forms of inheritance, and variants within the same gene often can be associated with multiple distinct phenotypes. Such aspects of the IRDs highlight the difficulty met when establishing a genetic diagnosis in patients. Here we provide an overview of cutting-edge next-generation sequencing techniques and strategies currently in use to maximise the effectivity of IRD gene screening. These techniques have helped researchers globally to find elusive causes of IRDs, including copy number variants, structural variants, new IRD genes and deep intronic variants, among others. Resolving a genetic diagnosis with thorough testing enables a more accurate diagnosis and more informed prognosis and should also provide information on inheritance patterns which may be of particular interest to patients of a child-bearing age. Given that IRDs are heritable conditions, genetic counselling may be offered to help inform family planning, carrier testing and prenatal screening. Additionally, a verified genetic diagnosis may enable access to appropriate clinical trials or approved medications that may be available for the condition

Multimodal imaging in a pedigree of X-linked Retinoschisis with a novel RS1 variant

Abstract Background To describe the clinical phenotype and genetic cause underlying the disease pathology in a pedigree (affected n = 9) with X-linked retinoschisis (XLRS1) due to a novel RS1 mutation and to assess suitability for novel therapies using multimodal imaging. Methods The Irish National Registry for Inherited Retinal Degenerations (Target 5000) is a program including clinical history and examination with multimodal retinal imaging, electrophysiology, visual field testing and genetic analysis. Nine affected patients were identified across 3 generations of an XLRS1 pedigree. DNA sequencing was performed for each patient, one carrier female and one unaffected relative. Pedigree mapping revealed a further 4 affected males. Results All affected patients had a history of reduced visual acuity and dyschromatopsia; however, the severity of phenotype varied widely between the nine affected subjects. The stage of disease was classified as previously described. Phenotypic severity was not linearly correlated with age. A novel RS1 (Xp22.2) mutation was detected (NM_000330: c.413C > A) resulting in a p.Thr138Asn substitution. Protein modelling demonstrated a change in higher order protein folding that is likely pathogenic. Conclusions This family has a novel gene mutation in RS1 with clinical evidence of XLRS1. A proportion of the older generation has developed end-stage macular atrophy; however, the severity is variable. Confirmation of genotype in the affected grandsons of this pedigree in principle may enable them to avail of upcoming gene therapies, provided there is anatomical evidence (from multimodal imaging) of potentially reversible early stage disease

A randomized controlled trial of two types of in-shoe orthoses in children with flexible excess pronation of the feet

BACKGROUND: Orthoses for children with flexible excess pronation are estimated to cost Australian parents millions of dollars per year; however, there is no high-level evidence that orthoses improve function or reduce pain. METHODS: A randomized parallel, single-blinded, controlled trial of custom-made and ready-made orthoses was conducted in children between the ages of 7 and 11 years with bilateral flexible excess pronation. The diagnosis was based on calcaneal eversion and navicular drop. Outcomes included gross motor proficiency, self-perception, exercise efficiency, and pain. Measurements were taken at baseline, and at 3 and 12 months. Of the 178 children who participated at baseline, 160 continued to the end of the trial. RESULTS: After randomization, baseline characteristics were similar between the three treatment groups (custom-made, ready-made, and control). Statistical modeling demonstrated that although for most outcome measures there were statistically significant trends over time, none of the group comparisons were statistically significant. A sub-group analysis of those presenting with pain found no significant differences at 3 or 12 months. CONCLUSIONS: This study found no evidence to justify the use of in-shoe orthoses in the management of flexible excess foot pronation in children

Target 5000: Target Capture Sequencing for Inherited Retinal Degenerations

There are an estimated 5000 people in Ireland who currently have an inherited retinal degeneration (IRD). It is the goal of this study, through genetic diagnosis, to better enable these 5000 individuals to obtain a clearer understanding of their condition and improved access to potentially applicable therapies. Here we show the current findings of a target capture next-generation sequencing study of over 750 patients from over 520 pedigrees currently situated in Ireland. We also demonstrate how processes can be implemented to retrospectively analyse patient datasets for the detection of structural variants in previously obtained sequencing reads. Pathogenic or likely pathogenic mutations were detected in 68% of pedigrees tested. We report nearly 30 novel mutations including three large structural variants. The population statistics related to our findings are presented by condition and credited to their respective candidate gene mutations. Rediagnosis rates of clinical phenotypes after genotyping are discussed. Possible causes of failure to detect a candidate mutation are evaluated. Future elements of this project, with a specific emphasis on structural variants and non-coding pathogenic variants, are expected to increase detection rates further and thereby produce an even more comprehensive representation of the genetic landscape of IRDs in Ireland

MFRP-Associated Retinopathy and Nanophthalmos in Two Irish Probands: A Case Report

The conjunction of nanophthalmos (NO) and retinitis pigmentosa (RP) provides challenges to effective clinical management while narrowing the genetic spectrum for targeted molecular diagnostics. This case study describes two not knowingly related adult cases of MFRP-associated retinopathy and nanophthalmos (MARN). Structural features including short axial lengths (mean 16.4 mm), steep keratometry (mean 49.98 D), adult-onset signs, and symptoms of retinal dystrophy and acquired disease (i.e., cataract, angle-closure glaucoma) were evident in both cases. Pathogenic variants in the MFRP gene impair both prenatal eye growth and childhood emmetropization while also leading to RPE/outer retinal degeneration in 75% of cases. We discuss the “small-eye” phenotype spectrum and associated defining characteristics, molecular mechanisms with particular focus on MFRP-associated NO with RP features (MARN), the spectrum of visual morbidities (e.g., extreme refractive error, amblyopia, cystoid macular lesions, early cataract) and the challenges of their treatment/surgical management