Molecular and clinical analysis of 27 German patients with Leber congenital amaurosis

Weisschuh, Nicole; Feldhaus, Britta; Khan, Muhammad Imran; Cremers, Frans P.M.; Kohl, Susanne; Wissinger, Bernd; Zobor, Ditta

doi:10.1371/journal.pone.0205380

Cited by 42 publications

(34 citation statements)

References 27 publications

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“…Genetic testing comprised candidate gene sequencing ( n = 1, ARRP171-15079), IRD gene panel sequencing ( n = 19), whole-genome sequencing ( n = 1, ARRP411-30491), or testing for familial variants by Sanger sequencing in a research and/or diagnostic genetic set-up [ 41 , 42 , 43 ]. A segregation analysis was performed on the parental samples available from six index cases to confirm a biallelic status of the identified PDE6B variants ( Table 1 ).…”

Section: Methodsmentioning

confidence: 99%

Clinical Phenotype of PDE6B-Associated Retinitis Pigmentosa

Kuehlewein

Zobor

Štingl

et al. 2021

IJMS

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In this retrospective, longitudinal, observational cohort study, we investigated the phenotypic and genotypic features of retinitis pigmentosa associated with variants in the PDE6B gene. Patients underwent clinical examination and genetic testing at a single tertiary referral center, including best-corrected visual acuity (BCVA), kinetic visual field (VF), full-field electroretinography, full-field stimulus threshold, spectral domain optical coherence tomography, and fundus autofluorescence imaging. The genetic testing comprised candidate gene sequencing, inherited retinal disease gene panel sequencing, whole-genome sequencing, and testing for familial variants by Sanger sequencing. Twenty-four patients with mutations in PDE6B from 21 families were included in the study (mean age at the first visit: 32.1 ± 13.5 years). The majority of variants were putative splicing defects (8/23) and missense (7/23) mutations. Seventy-nine percent (38/48) of eyes had no visual acuity impairment at the first visit. Visual acuity impairment was mild in 4% (2/48), moderate in 13% (6/48), and severe in 4% (2/48). BCVA was symmetrical in the right and left eyes. The kinetic VF measurements were highly symmetrical in the right and left eyes, as was the horizontal ellipsoid zone (EZ) width. Regarding the genetic findings, 43% of the PDE6B variants found in our patients were novel. Thus, this study contributed substantially to the PDE6B mutation spectrum. The visual acuity impairment was mild in 83% of eyes, providing a window of opportunity for investigational new drugs. The EZ width was reduced in all patients and was highly symmetric between the eyes, making it a promising outcome measure. We expect these findings to have implications on the design of future PDE6B-related retinitis pigmentosa (RP) clinical trials.

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Section: Methodsmentioning

confidence: 99%

Clinical Phenotype of PDE6B-Associated Retinitis Pigmentosa

Kuehlewein

Zobor

Štingl

et al. 2021

IJMS

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“…DNA concentration was assessed with a Quant-iT™ dsDNA Assay Kit, broad range, using a NanoDrop 3300 spectrofluorometer (Thermofisher Scientific, Waltham, MA, USA). Subsequently, samples were screened using a molecular inversion probes (MIPs) technique targeting 108 genes involved in the pathogenesis of inherited retinal disorders (IRDs), as described elsewhere [17]. Briefly, over 6000 MIPs covering the regions of interest were designed using an in-house software (Department of Human Genetics, Radboud University Nijmegen Medical Center) and manually verified.…”

Section: Methodsmentioning

confidence: 99%

Genetic Spectrum of ABCA4-Associated Retinal Degeneration in Poland

Tracewska

Kocyła-Karczmarewicz

Rafalska

et al. 2019

Genes

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Mutations in retina-specific ATP-binding cassette transporter 4 (ABCA4) are responsible for over 95% of cases of Stargardt disease (STGD), as well as a minor proportion of retinitis pigmentosa (RP) and cone-rod dystrophy cases (CRD). Since the knowledge of the genetic causes of inherited retinal diseases (IRDs) in Poland is still scarce, the purpose of this study was to identify pathogenic ABCA4 variants in a subgroup of Polish IRD patients. We recruited 67 families with IRDs as a part of a larger study. The patients were screened with next generation sequencing using a molecular inversion probes (MIPs)-based technique targeting 108 genes involved in the pathogenesis of IRDs. All identified mutations were validated and their familial segregation was tested using Sanger sequencing. In the case of the most frequent complex allele, consisting of two variants in exon 12 and 21, familial segregation was tested using restriction fragment length polymorphism (RFLP). The most prevalent variant, a complex change c.[1622T>C;3113C>T], p.[Leu541Pro;Ala1038Val], was found in this cohort in 54% of all solved ABCA4-associated disorder cases, which is the highest frequency reported thus far. Additionally, we identified nine families displaying a pseudo-dominant mode of inheritance, indicating a high frequency of pathogenic variants within this population.

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“…Due to their dual annealing properties, smMIPs and MIPs are very specific and have been used in multiplex PCR enrichment consisting of 1,312 or 6,200 probes to analyze 33 genes involved in cancer (Hiatt, Pritchard, Salipante, O'Roak, & Shendure, 2013) or 108 genes implicated in IRDs (Weisschuh et al, 2018), respectively. This technology requires an initial investment in synthesizing smMIP oligonucleotides and balancing of their targeting properties, but thereafter it is superior in terms of cost, throughput, scalability, sensitivity and specificity.…”

Section: Introductionmentioning

confidence: 99%

“…This technology requires an initial investment in synthesizing smMIP oligonucleotides and balancing of their targeting properties, but thereafter it is superior in terms of cost, throughput, scalability, sensitivity and specificity. smMIPs can be used for simultaneous sequencing of hundreds of patients (Neveling et al, 2017;Weisschuh et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…By employing smMIPs-based ABCA4 sequencing, a large number of cases could be simultaneously sequenced for variants in~11,500 bp, with an average coverage of 583×. In recent studies (Tayebi et al, 2019;Weisschuh et al, 2018), 647,547 bp was sequenced using 6,129 MIPs targeting 1,524 coding regions of 108 retinal dystrophy-associated genes, with an average coverage of 213x per MIP. This is comparable to other targeted sequencing methods such as WES or Haloplex-based sequencing.…”

Section: Deep-intronic Variants Identification and In Vitro Assessmentmentioning

confidence: 99%

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Cost‐effective molecular inversion probe‐based ABCA4 sequencing reveals deep‐intronic variants in Stargardt disease

et al. 2019

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Purpose Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation‐scanning methods. We aimed to develop a cost‐effective sequencing method for ABCA4 exons and regions carrying known causal deep‐intronic variants. Methods Fifty exons and 12 regions containing 14 deep‐intronic variants of ABCA4 were sequenced using double‐tiled single molecule Molecular Inversion Probe (smMIP)‐based next‐generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Results Thirty‐four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep‐intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep‐intronic variant (c.4539+2065C>G) resulted in a 170‐nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). Conclusions smMIPs‐based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost‐effective mutation detection in STGD1 cases in previously unsolved cases.

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Molecular and clinical analysis of 27 German patients with Leber congenital amaurosis

Cited by 42 publications

References 27 publications

Clinical Phenotype of PDE6B-Associated Retinitis Pigmentosa

Clinical Phenotype of PDE6B-Associated Retinitis Pigmentosa

Genetic Spectrum of ABCA4-Associated Retinal Degeneration in Poland

Cost‐effective molecular inversion probe‐based ABCA4 sequencing reveals deep‐intronic variants in Stargardt disease

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