Limitations of short-read NGS in detecting RP1 Alu insertions: a case emphasizing Sanger confirmation

Using Molecular Diagnostics for Inherited Retinal Dystrophies: The 6 "I"s That Are Necessary to Diagnose 2 Eyes Genetically.

Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 45% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations.

The purpose of this study was to screen Copy Number Variations (CNVs) in 35 unsolved Inherited Retinal Dystrophy (IRD) families. Initially, next generation sequencing, including a specific Hereditary Eye Disease Enrichment Panel or Whole exome sequencing, was employed to screen (likely) pathogenic Single-nucleotide Variants (SNVs) and small Insertions and Deletions (indels) for these cases. All available SNVs and indels were further validated and co-segregation analyses were performed in available family members by Sanger sequencing. If not, after excluding deep intronic variants, Multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescence PCR (QF-PCR) and Sanger sequencing were employed to screen CNVs. We determined that 18 probands who had heterozygous SNVs/indels or whose parents were not consanguineous but had homozygous SNVs/indels in autosomal recessive IRDs genes had CNVs in another allele of these genes, 11 families had disease-causing hemizygous CNVs in X-linked IRD genes, 6 families had (likely) pathogenic heterozygous CNVs in PRPF31 gene. Of 35 families, 33 different CNVs in 16 IRD-associated genes were detected, with PRPF31, EYS and USH2A the most common disease-causing gene in CNVs. Twenty-six and 7 of them were deletion and duplication CNVs, respectively. Among them, 14 CNVs were first reported in this study. Our research indicates that CNVs contribute a lot to IRDs, and screening of CNVs substantially increases the diagnostic rate of IRD. Our results emphasize that MLPA and QF-PCR are ideal methods to validate CNVs, and the novel CNVs reported herein expand the mutational spectrums of IRDs.

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.

Inherited retinal dystrophies (IRDs) are a group of clinically and genetically heterogeneous diseases involving more than 280 genes and no less than 20 different clinical phenotypes. In this study, our aims were to identify the disease-causing gene variants of 319 Chinese patients with IRD, and compare the pros and cons of targeted panel sequencing and whole exome sequencing (WES). Patients were assigned for analysis with a hereditary eye disease enrichment panel (HEDEP) or WES examination based on time of recruitment. This HEDEP was able to capture 441 hereditary eye disease genes, which included 291 genes related to IRD. As RPGR ORF15 was difficult to capture, all samples were subjected to Sanger sequencing for this region. Among the 163 disease-causing variants identified in this study, 73 had been previously reported, and the other 90 were novel. Genes most commonly implicated in different inheritances of IRDs in this cohort were presented. HEDEP and WES achieved diagnostic yield with 41.2% and 33.0%, respectively. In addition, nine patients were found to carry pathogenic mutations in the RPGR ORF15 region with Sanger sequencing. Our study demonstrates that HEDEP can be used as a first-tier test for patients with IRDs.

PurposeThis study was conducted to analyze the clinical features associated with the pathogenic variants of ABCA4 in Korean patients with inherited retinal dystrophies (IRDs).MethodsWe enrolled patients with IRDs who visited a tertiary referral hospital and identified the pathogenic variants of ABCA4 through targeted gene panel sequencing and whole exome sequencing. We analyzed the clinical characteristics and phenotypic spectrum according to genotype.ResultsEleven patients (from nine families) with IRDs and pathogenic variants in ABCA4 were included. Eight patients (from seven families) with Stargardt disease (STGD), two (from one family) with cone-rod dystrophy (CRD), and one with early-onset retinitis pigmentosa (RP) were included. Two heterozygous mutations were identified in eight families, and one variant was found in a patient with fundus flavimaculatus. Two variants, p.Gln294Ter and p.Gln636Lys, were associated with severe phenotypes, such as early-onset RP and CRD. Four novel pathogenic variants, p.Gln636Lys, p.Ile1114del, p.Thr1117Ala, and p.Asn1588Tyr, were identified. p.Gln294Ter, p.Leu1157Ter, and p.Lys2049ArgfsTer12 were repeatedly detected in Koreans with ABCA4-associated retinal diseases (ABCA4-RD).ConclusionsVarious pathogenic variants of ABCA4, including four novel variants, were identified, and ABCA4-RD exhibited various phenotypes and disease severities in a Korean IRD cohort. These findings will be useful for understanding the clinical features of ABCA4-RD and ethnicity-specific variants in East Asians.

Inherited retinal dystrophies (IRDs) are a global problem that is largely unaddressed, especially in Africa. Black indigenous Africans are rarely represented in research that develops genetic tests and genetic therapies for IRDs, yet their genomes are more diverse. The aim of this literature review is to synthesize information on the IRD genetic research conducted among indigenous black Africans to identify challenges and opportunities for progress. PubMed was searched to identify empirical publications reporting the genetic analysis of IRDs among indigenous Africans. A total of 11 articles were selected for the review. Based on the information in the articles, the main genetic testing methods in use include next-generation, whole exome, and Sanger sequencing. The main IRDs characterized by the genetic tests include retinitis pigmentosa, Leber Congenital Amaurosis, Stagardt disease, and cone dystrophy. Examples of implicated genes include MERTK, GUCY2D, ABCA4, and KCNV2 for the four IRDs, respectively. Research activities on the genetics of IRDs are generally scanty in Africa. Even in South Africa and North Africa where some research activities were noted, only a few indigenous black Africans were included in the study cohorts. There is an urgent need for genetic research on IRDs, especially in East, Central, and West Africa.

Whole Exome Sequencing (WES) is a powerful approach for detecting sequence variations in the human genome. The aim of this study was to investigate the genetic defects in Jordanian patients with inherited retinal dystrophies (IRDs) using WES. WES was performed on proband patients’ DNA samples from 55 Jordanian families. Sanger sequencing was used for validation and segregation analysis of the detected, potential disease-causing variants (DCVs). Thirty-five putatively causative variants (6 novel and 29 known) in 21 IRD-associated genes were identified in 71% of probands (39 of the 55 families). Three families showed phenotypes different from the typically reported clinical findings associated with the causative genes. To our knowledge, this is the largest genetic analysis of IRDs in the Jordanian population to date. Our study also confirms that WES is a powerful tool for the molecular diagnosis of IRDs in large patient cohorts.

Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel or next generation [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein-coding genes in the genome (≈1.1% of genome), and an exome test for a single patient generates ≈6 gigabases (109 bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ≈27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. Decreasing sequencing costs and broad dissemination of next-generation (next-gen) equipment and expertise are increasing availability of massively parallel sequencing of patient DNA samples (short read massively parallel or next-gen sequencing).1,2 Most rapidly expanding is exome sequencing, where all protein-coding sequences (exons) are selected from total genomic DNA and selectively sequenced.3 Alternative approaches to next-gen sequencing include targeted sequencing (TS) and whole genome (complete genome) sequencing. Currently, marketed targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel next-gen approaches are beginning to supplant …

Inherited Retinal Dystrophies (IRD) are rare and heterogeneous blindness-causing diseases caused by pathogenic variants in genes involved in retina function. Thus, the diagnosis in patients with IRD is complicated and late, and it can also be confused with other diseases. For this reason, exome sequencing is a tool for accurate diagnosis. In this work, the exome of 17 Mexican patients clinically evaluated with IRD at the Conde de Valenciana Institute was analyzed. The exome sequencing analyses indicated that 88% (15/17) of the patients suffer from IRD: seven have conerod disorders, two suffer Usher syndrome, three suffer retinitis pigmentosa, two suffer Stargardt disease, and one suffer corneal dystrophy. Within the cohort, there are cases with complex alleles, and one case with a homogeneous variant, previously reported as heterogeneous, including a case whose variant causes lipofuscinosis but with an impact on IRD. Therefore, exome sequencing is an effective great tool for diagnosing rare diseases.

PurposeThis study aimed to identify the underlying genetic cause(s) of inherited retinal dystrophy (IRD) in 12 families of Kuwaiti origin affected by macular dystrophy and four Spanish patients affected by...

Inherited retinal dystrophies (IRD) are a group of conditions resulting in visual impairments or blindness, due to the dysfunction of the retina. It affects 1/2000 individuals worldwide, and over 324 genes and 20 phenotypes are implicated in these pathologies. The most common form of IRD is Retinitis Pigmentosa, followed by Stargardt diseases, Leber congenital amaurosis and cone/cone-rod dystrophies. Each form of IRDs presents different clinical features. This study aims to broaden the clinical and genetic investigations of IRD patients in Morocco. Whole exome sequencing was performed on the probands of two unrelated Moroccan families with IRD phenotypes, followed by Sanger sequencing to evaluate the segregation of candidate variants within family members. WES revealed two homozygous pathogenic splice-variants in CABP4:c.800-2A > G and TTLL5:c.182-1G > T in the two families, and was confirmed by Sanger sequencing that revealed a second homozygous variant in TTLL5 c.182-5T > C in the second family. All parents were heterozygous. This study reports novel pathogenic variants in TTLL5 and CABP4 in patients with IRDs. These findings expand our knowledge of IRD causal genes in Moroccan patients.

Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin

Inherited retinal dystrophies (IRDs) present a challenge in clinical diagnostics due to their pronounced genetic heterogeneity. Despite advances in next-generation sequencing (NGS) technologies, a substantial portion of the genetic basis underlying IRDs remains elusive. Addressing this gap seems important for gaining insights into the genetic landscape of IRDs, which may help improve diagnosis and prognosis and develop targeted therapies in the future. To provide a clinical and molecular characterization of 6 patients with IRDs with biallelic disease-causing variants in a novel candidate IRD disease gene. This multicenter case series study included 6 patients with IRDs from 4 tertiary hospitals (in the US: National Eye Institute, National Institutes of Health Clinical Center; in the UK: Moorfields Eye Hospital, Royal Liverpool University Hospital, Birmingham Women's and Children's). Biallelic disease-causing variants in the novel candidate IRD disease gene, UBAP1L. Participants underwent comprehensive clinical ophthalmic assessments to characterize the features of retinal dystrophy. Exome and genome sequencing revealed candidate variants in the UBAP1L gene; no other plausible disease variants in known IRD genes were identified. A minigene assay provided functional insights for a noncanonical splice variant, and a knockout mouse model was used for in vivo functional elucidation. Four homozygous UBAP1L variants were identified in the affected individuals from 6 families, including 2 frameshift variants (c.710del and c.634_644del), 1 canonical splice variant (c.121-2A>C), and 1 noncanonical splice variant (c.910-7G>A), which was shown to cause aberrant splicing and frameshift in a minigene assay. Participants presented with retinal dystrophy including maculopathy, cone dystrophy, and cone-rod dystrophy. Single-cell RNA sequencing of the retina showed that human UBAP1L is highly expressed in both cones and retinal pigment epithelium, whereas mouse Ubap1l is highly expressed in cone cells only. Mice with truncation of the C-terminal SOUBA domain did not manifest retinal degeneration up to 15 months of age. Study results reveal clinical and genetic evidence that loss of UBAP1L function was associated with inherited retinopathy in humans. These findings hold promise for improved clinical diagnostics, prognosis, and the potential development of targeted therapies for individuals affected by IRDs.

Introduction: Inherited retinal dystrophies (IRDs) associated with more than 300 genes are a clinically and genetically heterogeneous group of retinal diseases. This study aimed to identify causative gene variants and molecular basis of Turkish patients with IRD. Methods: Whole-exome sequencing was performed in 28 unrelated patients. The potential pathogenicity of variants was evaluated using the American College of Medical Genetics variant interpretation guidelines, in silico prediction tools, published literature or Human Gene Mutation Database, and compatibility with inheritance patterns or known phenotypes. Results: Causative variants in 21 genes, including MERTK, SNRP200, MYO7A, AIPL1, RDH12, OTX2, ADGRV1, RPGRIP1, SPATA7, USH2A, MFSD8, CDHR1, EYS, CACNA1F, CNGA3, RDH5, TULP1, BBS2, BEST1, RS1, GUCY2D were detected in 26 (92.9%) of 28 patients. The most prevalent causative variants were observed MERTK (10.7% of cases), followed by CDHR1, AIPL1, RDH12, SPATA7, CNGA3, TULP1 (7.1% of cases, each). The most common variant type in this study was missense variants (53%), followed by frameshift (21%), nonsense (20%), and splice (6%). Twelve novel variants, 6 of frameshift and 6 of missense, were detected in ten genes. Retinitis pigmentosa was the most common phenotype followed by Leber congenital amaurosis. Conclusion: This study provides an overview of causative gene variants in Turkish patients with IRD. Variants identified in this study expand the variant spectrum of IRD genes. We believe it is essential to combine molecular and clinical data to diagnose IRD patients, especially with the emergence of therapeutic options.