A novel phenotype-guided genome analysis pipeline for variant discovery.

Purpose Inherited retinal dystrophies (IRDs) encompass a genetically and clinically heterogeneous group of disorders, with over 300 genes currently implicated. Early and precise genetic diagnosis is critical for advancing targeted gene therapies and personalized treatment strategies. This study aims to investigate genetic findings in IRDs using whole-exome sequencing (WES) in a cohort of affected individuals. Methods WES was performed on 34 unrelated probands diagnosed with diverse IRD subtypes. Variant analysis focused on identifying clinically relevant variants consistent with the patients’ phenotypes and inheritance patterns, emphasizing novel and rare variants. Results Clinically relevant variants consistent with the patients’ phenotypes and inheritance patterns were identified in 24 cases. Among these, four variants were novel: RP2 c.181_182del, CYP4V2 c.377T>G, RPGR c.1414G>A, and RPE65 c.267C>G. Additionally, we identified a patient with isolated IRD carrying a homozygous IFT81 c.1969C>T (p.Gln657Ter) variant, a gene typically associated with syndromic phenotypes. Furthermore, we reported a relatively rare presentation of ARSG-related Usher syndrome in a patient harboring a homozygous ARSG c.263G>T (p.Arg88Leu) alteration. Conclusions This study underscores the diagnostic power of WES in IRDs, revealing novel variants and supporting its integration into clinical practice to enhance early diagnosis and enable precision medicine.

Inherited retinal dystrophies (IRDs) are Mendelian diseases with tremendous genetic and phenotypic heterogeneity. Identification of the underlying genetic basis of these dystrophies is therefore challenging. In this study we employed whole exome sequencing (WES) in 11 families with IRDs and identified disease-causing variants in 8 of them. Sequence analysis of about 250 IRD-associated genes revealed 3 previously reported disease-associated variants in RHO, BEST1 and RP1. We further identified 5 novel pathogenic variants in RPGRIP1 (p.Ser964Profs*37), PRPF8 (p.Tyr2334Leufs*51), CDHR1 (p.Pro133Arg and c.439-17G>A) and PRPF31 (p.Glu183_Met193dup). In addition to confirming the power of WES in genetic diagnosis of IRDs, we document challenges in data analysis and show cases where the underlying genetic causes of IRDs were missed by WES and required additional techniques. For example, the mutation c.439-17G>A in CDHR1 would be rated unlikely applying the standard WES analysis. Only transcript analysis in patient fibroblasts confirmed the pathogenic nature of this variant that affected splicing of CDHR1 by activating a cryptic splice-acceptor site. In another example, a 33-base pair duplication in PRPF31 missed by WES could be identified only via targeted analysis by Sanger sequencing. We discuss the advantages and challenges of using WES to identify mutations in heterogeneous diseases like IRDs.

BackgroundNGS-based genetic diagnosis has completely revolutionized the human genetics field. In this study, we have aimed to identify new genes and mutations by Whole Exome Sequencing (WES) responsible for inherited retinal dystrophies (IRD).MethodsA cohort of 33 pedigrees affected with a variety of retinal disorders was analysed by WES. Initial prioritization analysis included around 300 IRD-associated genes. In non-diagnosed families a search for pathogenic mutations in novel genes was undertaken.ResultsGenetic diagnosis was attained in 18 families. Moreover, a plausible candidate is proposed for 10 more cases. Two thirds of the mutations were novel, including 4 chromosomal rearrangements, which expand the IRD allelic heterogeneity and highlight the contribution of private mutations. Our results prompted clinical re-evaluation of some patients resulting in assignment to a syndromic instead of non-syndromic IRD. Notably, WES unveiled four new candidates for non-syndromic IRD: SEMA6B, CEP78, CEP250, SCLT1, the two latter previously associated to syndromic disorders. We provide functional data supporting that missense mutations in CEP250 alter cilia formation.ConclusionThe diagnostic efficiency of WES, and strictly following the ACMG/AMP criteria is 55% in reported causative genes or functionally supported new candidates, plus 30% families in which likely pathogenic or VGUS/VUS variants were identified in plausible candidates. Our results highlight the clinical utility of WES for molecular diagnosis of IRD, provide a wider spectrum of mutations and concomitant genetic variants, and challenge our view on syndromic vs non-syndromic, and causative vs modifier genes.

In this study, patients with inherited retinal dystrophies (IRDs) who visited Ningxia Eye Hospital from January 2015 to September 2023 were analyzed. Through Whole Exome Sequencing (WES) and Sanger verification, 17 probands carrying homozygous variants were detected. The association between the genotype and clinical phenotype of patients with homozygous variants was analyzed. Among all the patients, 3 patients (17.6%) had a family history of consanguineous marriage, and the onset age of 5 patients(29.4%) was less than 10 years. According to 12 patients (70.6% ), they had the best corrected visual acuity (monocular) < 0.3. 3 were blind, 9 with moderate to severe visual impairment, and 2 with mild visual impairment. 16 homozygous variants were detected in 9 different genes, of which 7 were novel homozygous variants, including frameshift variants, missense variants, and a copy number variant. These variants are related to clinical phenotypes such as Usher syndrome type II (USH2), Stargardt disease (STGD), retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and Bardet-Biedl syndrome (BBS) respectively. The results of the study indicate that more than 80% of persons with homozygous variant originated from non-consanguineous families, emphasizing the significance of genetic screening for individuals who lack a family history of consanguineous marriage and no obvious clinical phenotypes, but who may carry genetic pathogenic variants for genetic diseases. Furthermore, by analyzing the genotypes and clinical phenotypes of IRD patients from these 17 Chinese families, we have expanded the spectrum of variants in known pathogenic genes for IRDs and the range of clinical phenotypes associated with variants in these genes. We have identified couples at high risk of having affected offspring and individuals with moderate to severe IRDs, providing a basis for genetic counseling, reproductive decision-making, disease prevention, and management. Our findings highlight the association between homozygous variants and more severe clinical phenotypes within these families, thus laying the groundwork for future genetic screening and intervention strategies.

Aims/Purpose: Inherited retinal dystrophies (IRD) are a group of predominantly monogenic disorders which have genetically heterogeneous origins and display wide clinical phenotypic heterogeneity. The current study aims to describe the clinical diagnoses, demographics, and genetic aetiology of patients in an adult cohort of Welsh patients to provide insight into the rates of genetic diagnosis of IRD. This will help inform patient prognosis and counselling in future clinical encounters.Methods: A comprehensive database of patients attending the tertiary referral clinic from 2011 to 2022 was compiled contemporaneously. Demographic data were collated from patients' clinical records. Subjects underwent genetic screening, including targeted gene sequencing, next‐generation sequencing‐based gene panel, or whole exome sequencing, to investigate the causative mutations.Results: A total of 365 probands (191 men and 174 women). The median age at which the patients developed visual symptoms related to IRD was 42 ± 17.7 years (range, 1–86 years). Retinitis pigmentosa (176; 48.2%), Macular Dystrophy (49; 13.4%), and Cone Rod Dystrophy (41; 11.2%) were the most common clinical diagnoses in this cohort. Among all the subjects, a genetic mutation was identified in 193 (52.9%) patients and the genetic aetiology of diseases was confirmed in 147 (40.3%) patients. Pathogenic mutations were identified in 48 retinal genes with the most commonly affected genes being ABCA4, USH2A and GUCY2D with 78, 44 and 13 mutations identified overall.Conclusions: The establishment of a molecular genetic diagnosis in this group of patients serves as a basis for genetic counselling and will allow patients to enrol in current and future gene‐based clinical trials, and benefit from any future gene‐based treatments.

Inherited retinal dystrophies (IRDs) affect 1 in 3000 individuals worldwide and are genetically heterogeneous, with over 270 identified genes and loci; however, there are still many identified disorders with no current genetic etiology. Whole exome sequencing (WES) provides a hypothesis-free first examination of IRD patients in either a clinical or research setting to identify the genetic cause of disease. We present a study of IRD in ten families from Alberta, Canada, through the lens of novel gene discovery. We identify the genetic etiology of IRDs in three of the families to be variants in known disease-associated genes, previously missed by clinical investigations. In addition, we identify two potentially novel associations: LRP1 in early-onset drusen formation and UBE2U in a multi-system condition presenting with retinoschisis, cataracts, learning disabilities, and developmental delay. We also describe interesting results in our unsolved cases to provide further information to other investigators of these blinding conditions.

During the last 20years, our group has focused on identifying the genes and mutations causative of inherited retinal dystrophies (IRDs). By applying massive sequencing approaches (NGS) in more than 500 familial and sporadic cases, we attained high diagnostic efficiency (85%) with a custom target gene panel and over 75% using whole exome sequencing (WES). Close to 40% of pathogenic alleles are novel mutations, which demand specific in silico tests and in vitro assays. Notably, missense variants are by far the most common type of mutation identified (around 40%), with small in-frame indels being less frequent (2%). To fill the gap of unsolved cases, when no candidate gene or only a single pathogenic allele has been identified, additional scientific and technical issues remain to be addressed. Reliable detection of genomic rearrangements and copy number variants (partial or complete), deep intronic mutations, variants that cause aberrant splicing events in retina-specific transcripts, functional assessment of hypomorphic missense alleles, mutations in regulatory sequences, the contribution of modifier genes to the IRD phenotype, and detection of low heteroplasmy mtDNA mutations are among the new challenges to be met.

Nanopore sequencing is a widely-used high-throughput genome sequencing technology that can sequence long fragments of a genome into raw electrical signals at low cost. Nanopore sequencing requires two computationally-costly processing steps for accurate downstream genome analysis. The first step, basecalling, translates the raw electrical signals into nucleotide bases (i.e., A, C, G, T). The second step, read mapping, finds the correct location of a read in a reference genome. In existing genome analysis pipelines, basecalling and read mapping are executed separately. We observe in this work that such separate execution of the two most time-consuming steps inherently leads to ❨1❩ significant data movement and ❨2❩ redundant computations on the data, slowing down the genome analysis pipeline. This paper proposes GenPIP, an in-memory genome analysis accelerator that tightly integrates basecalling and read mapping. GenPIP improves the petformance of the genome analysis pipeline with two key mechanisms: ❨1❩ in-memory fine-grained collaborative execution of the major genome analysis steps in parallel}; ❨2❩ a new technique for early-rejection of low-quality and unmapped reads to timely stop the execution of genome analysis for such reads, reducing inefficient computation. Our experiments show that, for the execution of the genome analysis pipeline, GenPIP provides 41.6$\times$ (8.4$\times$) speedup and 32.8$\times$ (20.8$\times$) energy savings with negligible accuracy loss compared to the state-of-the-art software genome analysis tools executed on a state-of-the-art CPU (GPU). Compared to a design that combines state-of-the-art in-memory basecalling and read mapping accelerators, GenPIP provides 1.39$\times$ speedup and 1.37$\times$ energy savings.

Rapid genetic diagnosis in single‐gene movement disorders

Genome sequencing and analysis allow researchers to decode the functional information hidden in DNA sequences as well as to study cell to cell variation within a cell population. Traditionally, the primary bottleneck in genomic analysis pipelines has been the sequencing itself, which has been much more expensive than the computational analyses that follow. However, an important consequence of the continued drive to expand the throughput of sequencing platforms at lower cost is that often the analytical pipelines are struggling to keep up with the sheer amount of raw data produced. Computational cost and efficiency have thus become of ever increasing importance. Recent methodological advances, such as data sketching, accelerators and domain-specific libraries/languages, promise to address these modern computational challenges. However, despite being more efficient, these innovations come with a new set of trade-offs, both expected, such as accuracy versus memory and expense versus time, and more subtle, including the human expertise needed to use non-standard programming interfaces and set up complex infrastructure. In this Review, we discuss how to navigate these new methodological advances and their trade-offs.

Genetic counseling in direct-to-consumer exome sequencing: a case report.

ACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG)

BackgroundLess than two percent of the human genome is protein coding, yet that small fraction harbours the majority of known disease causing mutations. Despite rapidly falling whole genome sequencing (WGS) costs, much research and increasingly the clinical use of sequence data is likely to remain focused on the protein coding exome. We set out to quantify and understand how WGS compares with the targeted capture and sequencing of the exome (exome-seq), for the specific purpose of identifying single nucleotide polymorphisms (SNPs) in exome targeted regions.ResultsWe have compared polymorphism detection sensitivity and systematic biases using a set of tissue samples that have been subject to both deep exome and whole genome sequencing. The scoring of detection sensitivity was based on sequence down sampling and reference to a set of gold-standard SNP calls for each sample. Despite evidence of incremental improvements in exome capture technology over time, whole genome sequencing has greater uniformity of sequence read coverage and reduced biases in the detection of non-reference alleles than exome-seq. Exome-seq achieves 95% SNP detection sensitivity at a mean on-target depth of 40 reads, whereas WGS only requires a mean of 14 reads. Known disease causing mutations are not biased towards easy or hard to sequence areas of the genome for either exome-seq or WGS.ConclusionsFrom an economic perspective, WGS is at parity with exome-seq for variant detection in the targeted coding regions. WGS offers benefits in uniformity of read coverage and more balanced allele ratio calls, both of which can in most cases be offset by deeper exome-seq, with the caveat that some exome-seq targets will never achieve sufficient mapped read depth for variant detection due to technical difficulties or probe failures. As WGS is intrinsically richer data that can provide insight into polymorphisms outside coding regions and reveal genomic rearrangements, it is likely to progressively replace exome-seq for many applications.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2105-15-247) contains supplementary material, which is available to authorized users.

Intellectual disability (ID) is a group of neurodevelopmental disorders with high heterogeneous in both genotypes and phenotypes and its definitive diagnosis is increasingly dependent ongenome-wide molecular diagnostics.Based on next generation sequencing(NGS), panel sequencing, whole exome sequencing (WES) and even whole genome sequencing are well applied to the molecular diagnosis of ID. Based on these, we recommend WES, especially trio-WES as the preferred detection method. NGS data analysis and reanalysis for ID have clinical significance for diagnosis, and can detect small scale variation and copy number variation in the genome reliably. Therefore, it has the potential to become the next recommended molecular diagnostic toolfor ID. Key words: Intellectual disability; Next generation sequencing; Whole exome sequencing; Mutation

Objective To review recent advances in genetic diagnosis of sensorineural hearing loss (SNHL) using gene panels, exome, and genome sequencing. Design A scoping review. Articles published from January 2022 to May 2024 on gene panels, exome, or genome sequencing for early SNHL diagnosis were reviewed. Study sample After removing duplicates and abstract and full-text review, 26 articles met inclusion criteria. Results Approximately, 60% of SNHL cases are genetic, with over 148 genes linked to non-syndromic forms and 700–800 associated but unconfirmed. Next-generation sequencing (NGS) has transformed etiologic diagnosis, with yields up to 79% depending on test and population. Exome sequencing achieves 40–60% diagnostic accuracy for bilateral SNHL and 1–5% for unilateral, rising to 18.3% with syndromic cases. However, challenges persist in cost, test performance, and variant interpretation, with newborn screening potentially missing 25–30% of SNHL cases. Conclusions Findings underscore the need for optimised screening, robust variant interpretation frameworks, and personalised counselling. Diagnostic approaches should be tailored, with NGS gene panels or exome sequencing recommended for bilateral cases and unilateral cases after excluding non-genetic causes. Genome sequencing may be pursued if no genetic cause is found, with follow-up. Integrating genetic diagnostics into standard care could improve outcomes and interventions for SNHL patients.