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

2019 ◽  
Vol 40 (10) ◽  
pp. 1749-1759 ◽  
Author(s):  
Mubeen Khan ◽  
Stéphanie S. Cornelis ◽  
Muhammad Imran Khan ◽  
Duaa Elmelik ◽  
Eline Manders ◽  
...  
Keyword(s):  
Cost Effective ◽  
Stargardt Disease ◽  
Molecular Inversion Probe ◽  
Intronic Variants

scholarly journals Resolving the dark matter of ABCA4 for 1,054 Stargardt disease probands through integrated genomics and transcriptomics

2019 ◽  
Author(s):  
Mubeen Khan ◽  
Stéphanie S. Cornelis ◽  
Marta del Pozo-Valero ◽  
Laura Whelan ◽  
Esmee H. Runhart ◽  
...  
Keyword(s):  
Data Storage ◽  
Single Molecule ◽  
Large Scale ◽  
Cost Effective ◽  
Chromosome 1 ◽  
Stargardt Disease ◽  
Model Study ◽  
Integrated Genomics ◽  
Intronic Variants

ABSTRACTMissing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases. The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.

scholarly journals Prevalence of ABCA4 Deep-Intronic Variants and Related Phenotype in An Unsolved “One-Hit” Cohort with Stargardt Disease

2019 ◽  
Vol 20 (20) ◽  
pp. 5053 ◽  
Author(s):  
Nassisi ◽  
Mohand-Saïd ◽  
Andrieu ◽  
Antonio ◽  
Condroyer ◽  
...  
Keyword(s):  
In Silico ◽  
Cohort Analysis ◽  
Pathogenic Variant ◽  
Stargardt Disease ◽  
Phenotypic Analysis ◽  
Variable Effect ◽  
Mild Phenotype ◽  
Related Phenotype ◽  
Novel Variants ◽  
Intronic Variants

We investigated the prevalence of reported deep-intronic variants in a French cohort of 70 patients with Stargardt disease harboring a monoallelic pathogenic variant on the exonic regions of ABCA4. Direct Sanger sequencing of selected intronic regions of ABCA4 was conducted. Complete phenotypic analysis and correlation with the genotype was performed in case a known intronic pathogenic variant was identified. All other variants found on the analyzed sequences were queried for minor allele frequency and possible pathogenicity by in silico predictions. The second mutated allele was found in 14 (20%) subjects. The three known deep-intronic variants found were c.5196+1137G>A in intron 36 (6 subjects), c.4539+2064C>T in intron 30 (4 subjects) and c.4253+43G>A in intron 28 (4 subjects). Even though the phenotype depends on the compound effect of the biallelic variants, a genotype-phenotype correlation suggests that the c.5196+1137G>A was mostly associated with a mild phenotype and the c.4539+2064C>T with a more severe one. A variable effect was instead associated with the variant c.4253+43G>A. In addition, two novel variants, c.768+508A>G and c.859-245_859-243delinsTGA never associated with Stargardt disease before, were identified and a possible splice defect was predicted in silico. Our study calls for a larger cohort analysis including targeted locus sequencing and 3D protein modeling to better understand phenotype-genotype correlations associated with deep-intronic changes and patients’ selection for clinical trials.

scholarly journals Identification of Novel Mutations inABCA4Gene: Clinical and Genetic Analysis of Indian Patients with Stargardt Disease

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Rajani Battu ◽  
Anshuman Verma ◽  
Ramesh Hariharan ◽  
Shuba Krishna ◽  
Ravi Kiran ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Vision Loss ◽  
Variant Calling ◽  
Signs And Symptoms ◽  
Cost Effective ◽  
Compound Heterozygous ◽  
Homozygous Mutation ◽  
Stargardt Disease ◽  
Indian Patients ◽  
Targeted Gene Sequencing

Stargardt disease (STGD) is the leading cause of juvenile macular degeneration associated with progressive central vision loss, photophobia, and colour vision abnormalities. In this study, we have described the clinical and genetic features of Stargardt patients from an Indian cohort. The next generation sequencing was carried out in five clinically confirmed unrelated patients and their family members using a gene panel comprising 184 retinal specific genes. Sequencing results were analyzed by read mapping and variant calling in genes of interest, followed by their verification and interpretation. Genetic analysis revealedABCA4mutations in all of the five unrelated patients. Among these, four patients were found with compound heterozygous mutations and another one had homozygous mutation. All the affected individuals showed signs and symptoms consistent with the disease phenotype. We report two novelABCA4mutations in Indian patients with STGD disease, which expands the existing spectrum of disease-causing variants and the understanding of phenotypic and genotypic correlations. Screening for causative mutations in patients with STGD using panel of targeted gene sequencing by NGS would be a cost effective tool, might be helpful in confirming the precise diagnosis, and contributes towards the genetic counselling of asymptomatic carriers and isolated patients.

scholarly journals Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4

2021 ◽  
Vol 22 (9) ◽  
pp. 4621
Author(s):  
Tomasz Z. Tomkiewicz ◽  
Nuria Suárez-Herrera ◽  
Frans P. M. Cremers ◽  
Rob W. J. Collin ◽  
Alejandro Garanto
Keyword(s):  
Antisense Oligonucleotide ◽  
Mrna Splicing ◽  
Stargardt Disease ◽  
Aberrant Splicing ◽  
Suitable Candidate ◽  
Pathogenic Variants ◽  
Splice System ◽  
Splicing Defects ◽  
Intronic Variants

The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.

scholarly journals Extremely hypomorphic and severe deep intronic variants in the ABCA4 locus result in varying Stargardt disease phenotypes

2018 ◽  
Vol 4 (4) ◽  
pp. a002733 ◽  
Author(s):  
Jana Zernant ◽  
Winston Lee ◽  
Takayuki Nagasaki ◽  
Frederick T. Collison ◽  
Gerald A. Fishman ◽  
...  
Keyword(s):  
Stargardt Disease ◽  
Disease Phenotypes ◽  
Intronic Variants

scholarly journals ATP-binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461-10T>C cause Stargardt disease due to defective splicing

2018 ◽  
Vol 96 (7) ◽  
pp. 737-743 ◽  
Author(s):  
Frida Jonsson ◽  
Ida Maria Westin ◽  
Lennart Österman ◽  
Ola Sandgren ◽  
Marie Burstedt ◽  
...  
Keyword(s):  
Atp Binding ◽  
Stargardt Disease ◽  
Intronic Variants ◽  
Atp Binding Cassette

scholarly journals Molecular Inversion Probe-Based Sequencing of USH2A Exons and Splice Sites as a Cost-Effective Screening Tool in USH2 and arRP Cases

2021 ◽  
Vol 22 (12) ◽  
pp. 6419
Author(s):  
Janine Reurink ◽  
Adrian Dockery ◽  
Dominika Oziębło ◽  
G. Jane Farrar ◽  
Monika Ołdak ◽  
...  
Keyword(s):  
Screening Tool ◽  
Usher Syndrome ◽  
Copy Number Variants ◽  
Genetic Diagnosis ◽  
Cost Effective ◽  
Single Nucleotide Variants ◽  
Effective Screening ◽  
Pathogenic Variants ◽  
Future Therapy ◽  
Molecular Inversion Probe

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

The application of electron microscopy to diagnosis in gynecologic neoplasms and tumor-like conditions

1984 ◽  
Vol 42 ◽  
pp. 102-105
Author(s):  
Lawrence M. Roth
Keyword(s):  
Electron Microscopy ◽  
Reproductive Tract ◽  
Malignant Tumors ◽  
Frozen Section ◽  
Cost Effective ◽  
Female Reproductive Tract ◽  
Ultrastructural Examination ◽  
Specific Diagnosis ◽  
Potential Value ◽  
Gynecologic Neoplasms

The female reproductive tract may be the site of a wide variety of benign and malignant tumors, as well as non-neoplastic tumor-like conditions, most of which can be diagnosed by light microscopic examination including special stains and more recently immunoperoxidase techniques. Nevertheless there are situations where ultrastructural examination can contribute substantially to an accurate and specific diagnosis. It is my opinion that electron microscopy can be of greatest benefit and is most cost effective when applied in conjunction with other methodologies. Thus, I have developed an approach which has proved useful for me and may have benefit for others. In cases where it is deemed of potential value, glutaraldehyde-fixed material is obtained at the time of frozen section or otherwise at operation. Coordination with the gynecologic oncologist is required in the latter situation. This material is processed and blocked and is available if a future need arises.

The IBM-PC in electron microscopy

1996 ◽  
Vol 54 ◽  
pp. 612-613
Author(s):  
James F. Mancuso
Keyword(s):  
Head Start ◽  
Image Acquisition ◽  
Cost Effective ◽  
Financial Applications ◽  
Number Of Factors ◽  
Instrument Control ◽  
The Cost ◽  
Training And Support ◽  
Control Image ◽  
Ibm Pc

IBM PC compatible computers are widely used in microscopy for applications ranging from control to image acquisition and analysis. The choice of IBM-PC based systems over competing computer platforms can be based on technical merit alone or on a number of factors relating to economics, availability of peripherals, management dictum, or simple personal preference.IBM-PC got a strong “head start” by first dominating clerical, document processing and financial applications. The use of these computers spilled into the laboratory where the DOS based IBM-PC replaced mini-computers. Compared to minicomputer, the PC provided a more for cost-effective platform for applications in numerical analysis, engineering and design, instrument control, image acquisition and image processing. In addition, the sitewide use of a common PC platform could reduce the cost of training and support services relative to cases where many different computer platforms were used. This could be especially true for the microscopists who must use computers in both the laboratory and the office.

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