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Non-Invasive Prenatal Diagnosis of Retinoblastoma Inheritance by Combined Targeted Sequencing Strategies.

ABSTRACT: Retinoblastoma, the most common childhood eye cancer, presents in two forms: heritable or sporadic. Heritable retinoblastoma is caused by a germline mutation in the RB1 gene. Early diagnosis of children at risk of inheriting an RB1 mutation is crucial to achieve optimal clinical outcome. Currently, the majority of genetic testing is performed on newborns, which has multiple disadvantages for both families and the healthcare system. We have developed a non-invasive prenatal diagnosis (NIPD) service for retinoblastoma, available from 8 weeks' gestation, which uses a combination of massively parallel sequencing (MPS) techniques, dependent on the inheritance model. Detection of paternal or suspected de novo RB1 variants is achieved through amplicon-based MPS. NIPD of a fetus at risk of maternal inheritance is performed using capture-based targeted sequencing and relative haplotype dosage analysis. In addition, we show proof of principle of how capture-based sequencing can be used for de novo variants unsuitable for amplicon-based testing. In total, we report the NIPD of 15 pregnancies, results of which show 100% concordance with all postnatal testing performed at the time of publication (n = 12) with remaining pregnancies ongoing. NIPD of retinoblastoma therefore offers a viable alternative to newborn genetic testing.

SUBMITTER: Gerrish A

PROVIDER: S-EPMC7692133 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Non-Invasive Prenatal Diagnosis of Retinoblastoma Inheritance by Combined Targeted Sequencing Strategies.

Gerrish Amy A Bowns Benjamin B Mashayamombe-Wolfgarten Chipo C Young Elizabeth E Court Samantha S Bott Joshua J McCalla Maureen M Ramsden Simon S Parks Michael M Goudie David D Carless Sue S Clokie Samuel S Cole Trevor T Allen Stephanie S

Journal of clinical medicine 20201030 11

Retinoblastoma, the most common childhood eye cancer, presents in two forms: heritable or sporadic. Heritable retinoblastoma is caused by a germline mutation in the <i>RB1</i> gene. Early diagnosis of children at risk of inheriting an <i>RB1</i> mutation is crucial to achieve optimal clinical outcome. Currently, the majority of genetic testing is performed on newborns, which has multiple disadvantages for both families and the healthcare system. We have developed a non-invasive prenatal diagnosi ...[more]

PMID: 33143217

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Project description:BackgroundSince the discovery of cell-free foetal DNA in the plasma of pregnant women, many non-invasive prenatal testing assays have been developed. In the area of skeletal dysplasia diagnosis, some PCR-based non-invasive prenatal testing assays have been developed to facilitate the ultrasound diagnosis of skeletal dysplasias that are caused by de novo mutations. However, skeletal dysplasias are a group of heterogeneous genetic diseases, the PCR-based method is hard to detect multiple gene or loci simultaneously, and the diagnosis rate is highly dependent on the accuracy of the ultrasound diagnosis. In this study, we investigated the feasibility of using targeted capture sequencing to detect foetal de novo pathogenic mutations responsible for skeletal dysplasia.Methodology/principal findingsThree families whose foetuses were affected by skeletal dysplasia and two control families whose foetuses were affected by other single gene diseases were included in this study. Sixteen genes related to some common lethal skeletal dysplasias were selected for analysis, and probes were designed to capture the coding regions of these genes. Targeted capture sequencing was performed on the maternal plasma DNA, the maternal genomic DNA, and the paternal genomic DNA. The de novo pathogenic variants in the plasma DNA data were identified using a bioinformatical process developed for low frequency mutation detection and a strict variant interpretation strategy. The causal variants could be specifically identified in the plasma, and the results were identical to those obtained by sequencing amniotic fluid samples. Furthermore, a mean of 97% foetal specific alleles, which are alleles that are not shared by maternal genomic DNA and amniotic fluid DNA, were identified successfully in plasma samples.Conclusions/significanceOur study shows that capture sequencing of maternal plasma DNA can be used to non-invasive detection of de novo pathogenic variants. This method has the potential to be used to facilitate the prenatal diagnosis of skeletal dysplasia.

Project description:Background Skeletal dysplasia (SD) is one of the most common inherited neonatal disorders worldwide, where the recurrent pathogenic mutations in the FGFR2, FGFR3, COL1A1, COL1A2 and COL2A1 genes are frequently reported in both non-lethal and lethal SD. The traditional prenatal diagnosis of SD using ultrasonography suffers from lower accuracy and performed at latter gestational stage. Therefore, it remains in desperate need of precise and accurate prenatal diagnosis of SD in early pregnancy. With the advancements of next-generation sequencing (NGS) technology and bioinformatics analysis, it is feasible to develop a NGS-based assay to detect genetic defects in association with SD in the early pregnancy. Methods An ampliseq-based targeted sequencing panel was designed to cover 87 recurrent hotspots reported in 11 common dominant SD and run on both Ion Proton and NextSeq550 instruments. Thirty-six cell-free and 23 genomic DNAs were used for assay developed. Spike-in DNA prepared from standard sample harboring known mutation and normal sample were also employed to validate the established SD workflow. Overall performances of coverage, uniformity, and on-target rate, and the detecting limitations on percentage of fetal fraction and read depth were evaluated. Results The established targeted-seq workflow enables a single-tube multiplex PCR for library construction and shows high amplification efficiency and robust reproducibility on both Ion Proton and NextSeq550 platforms. The workflow reaches 100% coverage and both uniformity and on-target rate are > 96%, indicating a high quality assay. Using spike-in DNA with different percentage of known FGFR3 mutation (c.1138 G > A), the targeted-seq workflow demonstrated the ability to detect low-frequency variant of 2.5% accurately. Finally, we obtained 100% sensitivity and 100% specificity in detecting target mutations using established SD panel. Conclusions An expanded panel for rapid and cost-effective genetic detection of SD has been developed. The established targeted-seq workflow shows high accuracy to detect both germline and low-frequency variants. In addition, the workflow is flexible to be conducted in the majority of the NGS instruments and ready for routine clinical application. Taken together, we believe the established panel provides a promising diagnostic or therapeutic strategy for prenatal genetic testing of SD in routine clinical practice. Supplementary Information The online version contains supplementary material available at 10.1186/s12920-021-01063-1.

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/

Dataset Information

Non-Invasive Prenatal Diagnosis of Retinoblastoma Inheritance by Combined Targeted Sequencing Strategies.

Publications

Non-Invasive Prenatal Diagnosis of Retinoblastoma Inheritance by Combined Targeted Sequencing Strategies.

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