Next Generation Sequencing targeted approach for the molecular diagnosis of Retinoblastoma.
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REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR861304_1.fastq.gz | Fastqsanger.gz | |||
| ERR861304_2.fastq.gz | Fastqsanger.gz | |||
| ERR861305_1.fastq.gz | Fastqsanger.gz | |||
| ERR861305_2.fastq.gz | Fastqsanger.gz | |||
| ERR861306_1.fastq.gz | Fastqsanger.gz |
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Project description:Retinoblastoma (RB, OMIM:180200) is the most common malignant childhood tumor of the eye with an estimated incidence between 1 in 16,000 and 1 in 18,000 live births [1,2]. RB is the first disease for which a genetic etiology of cancer has been described [3] being caused by mutations in the first tumor suppressor gene identified (RB1, Genbank accession # L11910). Mutations in both alleles of the RB1 gene are required for the development of this neoplasm [4], and, depending on the germ-line or somatic origin of the defect, a heritable or sporadic form can be distinguished. RB is unilateral in 60% of cases and only 15% of these are heritable [5]; in contrast, 40% of retinoblastomas are bilateral with risk of transmission to the offspring. Heritable retinoblastoma constitutes a cancer predisposition syndrome [6]. RB1 is located on chromosome 13 at band q14 and can be affected by a heterogeneous spectrum of genetic abnormalities, including chromosome translocation/deletion, genomic rearrangements, ranging from whole gene microdeletion to intragenic exons loss or duplication, and more than 900 different point mutations [7]. Mutational analysis is performed to search for the predisposing RB1 gene mutation in peripheral blood of patients with RB, but the molecular diagnosis requires several technical approaches to cover the entire field of oncogenic RB1 defects, frequently resulting in numerous, expensive and time consuming procedures. In particular, cytogenetic tools, such as classical chromosome investigations and Fluorescent In Situ Hybridization (FISH), in addition to Multiplex Ligation-dependent Probe Amplification (MLPA) technique, may account for detection of about 16% of RB1 abnormalities [8], while the remaining large amount of point mutations need to be investigated using sequencing analysis. Since the 1970s, Sanger sequencing has been recognized as the gold standard for mutation analysis in molecular diagnostics; however, its low-throughput, long turnaround time and overall cost [9] have called for new paradigms. Next Generation Sequencing (NGS) can massively sequence millions of DNA segments, promising low costs, increased workflow speed and enhanced sensitivity in mutation detection [9,10,11] On the other hand, conventional and molecular cytogenetic analysis, have been replaced by modern high-throughput investigations, such as array Comparative Genomic Hybridization (aCGH), that can reveal and measure cryptic genomic imbalances. In addition, aCGH can be focused on specific DNA segments or genes maximizing the resolution via a customized process. Based on these observations, we have recruited a cohort of retinoblastoma patients we previously investigated with conventional cytogenetics and MLPA. Patients diagnosed with RB but negative to the above standard screening have been tested with NGS to assess its ability in identifying RB causative mutations. On the other hand, patients positive to standard screening have been further investigated with RB1-custom array CGH analysis to characterize the genomic rearrangements with a better resolution compared to the conventional techniques. The complementary aCGH data set related to this study has also been deposited at ArrayExpress, under accession number E-MTAB-3492: https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3492/
2015-10-31 | E-MTAB-3515 | biostudies-arrayexpress
Project description:Background: Retinoblastoma (RB) is the most common malignant childhood tumor of the eye and results from inactivation of both alleles of the RB1 gene. Nowadays RB genetic diagnosis requires classical chromosome investigations, Multiplex Ligation-dependent Probe Amplification analysis (MLPA) and Sanger sequencing. Nevertheless, these techniques show some limitations. We report our experience on a cohort of RB patients using a combined approach of Next-Generation Sequencing (NGS) and RB1 custom array-Comparative Genomic Hybridization (aCGH). Methods: A total of 65 patients with retinoblastoma were studied: 29 cases of bilateral RB and 36 cases of unilateral RB. All patients were previously tested with conventional cytogenetics and MLPA techniques. Fifty-three samples were then analysed using NGS. Eleven cases were analysed by RB1 custom aCGH. One last case was studied only by classic cytogenetics. Finally, it has been tested, in a lab sensitivity assay, the capability of NGS to detect artificial mosaicism series in previously recognized samples prepared at 3 different mosaicism frequencies: 10%, 5%, 1%. Results: Of the 29 cases of bilateral RB, 28 resulted positive (96.5%) to the genetic investigation: 22 point mutations and 6 genomic rearrangements (four intragenic and two macrodeletion). A novel germline intragenic duplication, from exon18 to exon 23, was identified in a proband with bilateral RB. Of the 36 available cases of unilateral RB, 8 patients resulted positive (22%) to the genetic investigation: 3 patients showed point mutations while 5 carried large deletion. Finally, we successfully validated, in a lab sensitivity assay, the capability of NGS to accurately measure level of artificial mosaicism down to 1%. Conclusions: NGS and RB1-custom aCGH have demonstrated to be an effective combined approach in order to optimize the overall diagnostic procedures of RB. Custom aCGH is able to accurately detect genomic rearrangements allowing the characterization of their extension. NGS is extremely accurate in detecting single nucleotide variants, relatively simple to perform, cost savings and efficient and has confirmed a high sensitivity and accuracy in identifying low levels of artificial mosaicisms.
2015-10-31 | E-MTAB-3492 | biostudies-arrayexpress
Project description:Retinoblastoma (RB) is the most common malignant childhood tumor of the eye and results from inactivation of both alleles of the RB1 gene. Nowadays RB genetic diagnosis requires classical chromosome investigations, Multiplex Ligation-dependent Probe Amplification analysis (MLPA) and Sanger sequencing. Nevertheless, these techniques show some limitations. We report our experience on a cohort of RB patients using a combined approach of Next-Generation Sequencing (NGS) and RB1 custom array-Comparative Genomic Hybridization (aCGH).A total of 65 patients with retinoblastoma were studied: 29 cases of bilateral RB and 36 cases of unilateral RB. All patients were previously tested with conventional cytogenetics and MLPA techniques. Fifty-three samples were then analysed using NGS. Eleven cases were analysed by RB1 custom aCGH. One last case was studied only by classic cytogenetics. Finally, it has been tested, in a lab sensitivity assay, the capability of NGS to detect artificial mosaicism series in previously recognized samples prepared at 3 different mosaicism frequencies: 10, 5, 1 %.Of the 29 cases of bilateral RB, 28 resulted positive (96.5 %) to the genetic investigation: 22 point mutations and 6 genomic rearrangements (four intragenic and two macrodeletion). A novel germline intragenic duplication, from exon18 to exon 23, was identified in a proband with bilateral RB. Of the 36 available cases of unilateral RB, 8 patients resulted positive (22 %) to the genetic investigation: 3 patients showed point mutations while 5 carried large deletion. Finally, we successfully validated, in a lab sensitivity assay, the capability of NGS to accurately measure level of artificial mosaicism down to 1 %.NGS and RB1-custom aCGH have demonstrated to be an effective combined approach in order to optimize the overall diagnostic procedures of RB. Custom aCGH is able to accurately detect genomic rearrangements allowing the characterization of their extension. NGS is extremely accurate in detecting single nucleotide variants, relatively simple to perform, cost savings and efficient and has confirmed a high sensitivity and accuracy in identifying low levels of artificial mosaicisms.
| S-EPMC4632486 | biostudies-literature
Project description:BackgroundThe spectrum of RB1gene mutations in Retinoblastoma (RB) patients and the necessity of multiple traditional methods for complete variant analysis make the molecular diagnosis a cumbersome, labor-intensive and time-consuming process. Here, we have used targeted next generation sequencing (NGS) approach with in-house analysis pipeline to explore its potential for the molecular diagnosis of RB.MethodsThirty-three patients with RB and their family members were selected randomly. DNA from patient blood and/or tumor was used for RB1 gene targeted sequencing. The raw reads were obtained from Illumina Miseq. An in-house bioinformatics pipeline was developed to detect both single nucleotide variants (SNVs) and small insertions/deletions (InDels) and to distinguish between somatic and germline mutations. In addition, ExomeCNV and Cn. MOPS were used to detect copy number variations (CNVs). The pathogenic variants were identified with stringent criteria, and were further confirmed by conventional methods and cosegregation in families.ResultsUsing our approach, an array of pathogenic variants including SNVs, InDels and CNVs were detected in 85% of patients. Among the variants detected, 63% were germline and 37% were somatic. Interestingly, nine novel pathogenic variants (33%) were also detected in our study.ConclusionsWe demonstrated for the first time that targeted NGS is an efficient approach for the identification of wide spectrum of pathogenic variants in RB patients. This study is helpful for the molecular diagnosis of RB in a comprehensive and time-efficient manner.
| S-EPMC4415345 | biostudies-literature
Project description:Advantages of a Next Generation Sequencing targeted approach for the molecular diagnosis of Retinoblastoma.
| PRJEB9050 | ENA
Project description:BackgroundUsher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and time-consuming. Consequently, the aim of the present study was to develop a molecular diagnostics method for Usher syndrome, based on targeted next generation sequencing.MethodsA custom HaloPlex panel for Illumina platforms was designed to capture all exons of the 10 known causative Usher syndrome genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31 and CLRN1), the two Usher syndrome-related genes (HARS and PDZD7) and the two candidate genes VEZT and MYO15A. A cohort of 44 patients suffering from Usher syndrome was selected for this study. This cohort was divided into two groups: a test group of 11 patients with known mutations and another group of 33 patients with unknown mutations.ResultsForty USH patients were successfully sequenced, 8 USH patients from the test group and 32 patients from the group composed of USH patients without genetic diagnosis. We were able to detect biallelic mutations in one USH gene in 22 out of 32 USH patients (68.75%) and to identify 79.7% of the expected mutated alleles. Fifty-three different mutations were detected. These mutations included 21 missense, 8 nonsense, 9 frameshifts, 9 intronic mutations and 6 large rearrangements.ConclusionsTargeted next generation sequencing allowed us to detect both point mutations and large rearrangements in a single experiment, minimizing the economic cost of the study, increasing the detection ratio of the genetic cause of the disease and improving the genetic diagnosis of Usher syndrome patients.
| S-EPMC4245769 | biostudies-literature
Project description:BackgroundCholestasis is a frequent and severe condition during childhood. Genetic cholestatic diseases represent up to 25% of pediatric cholestasis. Molecular analysis by targeted-capture next generation sequencing (NGS) has recently emerged as an efficient diagnostic tool. The objective of this study is to evaluate the use of NGS in children with cholestasis.MethodsChildren presenting cholestasis were included between 2015 and 2020. Molecular sequencing was performed by targeted capture of a panel of 34 genes involved in cholestasis and jaundice. Patients were classified into three categories: certain diagnosis; suggested diagnosis (when genotype was consistent with phenotype for conditions without any available OMIM or ORPHANET-number); uncertain diagnosis (when clinical and para-clinical findings were not consistent enough with molecular findings).ResultsA certain diagnosis was established in 169 patients among the 602 included (28.1%). Molecular studies led to a suggested diagnosis in 40 patients (6.6%) and to an uncertain diagnosis in 21 patients (3.5%). In 372 children (61.7%), no molecular defect was identified.ConclusionsNGS is a useful diagnostic tool in pediatric cholestasis, providing a certain diagnosis in 28.1% of the patients included in this study. In the remaining patients, especially those with variants of uncertain significance, the imputability of the variants requires further investigations.
| S-EPMC9140938 | biostudies-literature
Project description:Advances in next-generation sequencing (NGS) promise to facilitate diagnosis of inherited disorders. Although in research settings NGS has pinpointed causal alleles using segregation in large families, the key challenge for clinical diagnosis is application to single individuals. To explore its diagnostic use, we performed targeted NGS in 42 unrelated infants with clinical and biochemical evidence of mitochondrial oxidative phosphorylation disease. These devastating mitochondrial disorders are characterized by phenotypic and genetic heterogeneity, with more than 100 causal genes identified to date. We performed "MitoExome" sequencing of the mitochondrial DNA (mtDNA) and exons of ~1000 nuclear genes encoding mitochondrial proteins and prioritized rare mutations predicted to disrupt function. Because patients and healthy control individuals harbored a comparable number of such heterozygous alleles, we could not prioritize dominant-acting genes. However, patients showed a fivefold enrichment of genes with two such mutations that could underlie recessive disease. In total, 23 of 42 (55%) patients harbored such recessive genes or pathogenic mtDNA variants. Firm diagnoses were enabled in 10 patients (24%) who had mutations in genes previously linked to disease. Thirteen patients (31%) had mutations in nuclear genes not previously linked to disease. The pathogenicity of two such genes, NDUFB3 and AGK, was supported by complementation studies and evidence from multiple patients, respectively. The results underscore the potential and challenges of deploying NGS in clinical settings.
| S-EPMC3523805 | biostudies-literature
Project description:BackgroundRetinoblastoma is a rare intraocular malignancy and typically initiated by inactivating biallelic mutations of RB1 gene. Each year, ~ 8000 children worldwide are diagnosed for retinoblastoma. In high-income countries, patient survival is over 95% while low-income countries is ~ 30%.If disease is diagnosed early and treated in centers specializing in retinoblastoma, the survival might exceed 95% and many eyes could be safely treated and support a lifetime of good vision. In China, approximate 1100 newly diagnosed cases are expected annually and 28 hospitals covering 25 provinces established centers classified by expertise and resources for better treatment options and follow-up. Comparing with other province of eastern China, Yunnan province is remote geographically. This might result that healthcare staff have low awareness of the role of genetic testing in management and screening in families.MethodsThe patients with retinoblastoma were selected in Yunnan. DNA from blood was used for targeted gene sequencing. Then, an in-house bioinformatics pipeline was done to detect both single nucleotide variants and small insertions/deletions. The pathogenic mutations were identified and further confirmed by conventional methods and cosegregation in families.ResultsUsing our approach, targeted next generation sequencing was used to detect the mutation of these 12 probands. Bioinformatic predictions showed that nine mutations were found in our study and four were novel pathogenic variants in these nine mutations.ConclusionsIt's the first report to describe RB1 mutations in Yunnan children with retinoblastoma. This study would improve role of genetic testing for management and family screening.
| S-EPMC7682003 | biostudies-literature
Project description:PurposeInherited peripheral neuropathies (IPN) represent a large heterogenous group of hereditary diseases with more than 100 causative genes reported to date. In this context, targeted next-generation sequencing (NGS) offers the opportunity to screen all these genes with high efficiency in order to unravel the genetic basis of the disease. Here, we compare the diagnostic yield of targeted NGS with our previous gene by gene Sanger sequencing strategy. We also describe several novel likely pathogenic variants.Design and participantsWe have completed the targeted NGS of 81 IPN genes in a cohort of 123 unrelated patients affected with diverse forms of IPNs, mostly Charcot-Marie-Tooth disease (CMT): 23% CMT1, 52% CMT2, 9% distal hereditary motor neuropathy, 7% hereditary sensory and autonomic neuropathy and 6.5% intermediate CMT.ResultsWe have solved the molecular diagnosis in 49 of 123 patients (~40%). Among the identified variants, 26 variants were already reported in the literature. In our cohort, the most frequently mutated genes are respectively: MFN2, SH3TC2, GDAP1, NEFL, GAN, KIF5A and AARS. Panel-based NGS was more efficient in familial cases than in sporadic cases (diagnostic yield 49%vs19%, respectively). NGS-based search for copy number variations, allowed the identification of three duplications in three patients and raised the diagnostic yield to 41%. This yield is two times higher than the one obtained previously by gene Sanger sequencing screening. The impact of panel-based NGS screening is particularly important for demyelinating CMT (CMT1) subtypes, for which the success rate reached 87% (36% only for axonal CMT2).ConclusionNGS allowed to identify causal mutations in a shorter and cost-effective time. Actually, targeted NGS is a well-suited strategy for efficient molecular diagnosis of IPNs. However, NGS leads to the identification of numerous variants of unknown significance, which interpretation requires interdisciplinary collaborations between molecular geneticists, clinicians and (neuro)pathologists.
| S-EPMC6224714 | biostudies-literature