DEVELOPMENT OF INNOVATIVE MOLECULAR AND SINGLE-CELL PREIMPLANTATION GENETIC DIAGNOSTIC AND SCREENING TESTS FOR DETECTING CGG-REPEAT EXPANSIONS IN THE FMR1 GENE

Ph.DDOCTOR OF PHILOSOPHY (SOM

Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders

Fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability and autism. Molecular diagnostic testing of FXS and related disorders (fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS)) relies on a combination of polymerase chain reaction (PCR) and Southern blot (SB) for the fragile X mental retardation 1 (FMR1) CGG-repeat expansion and methylation analyses. Recent advancements in PCR-based technologies have enabled the characterization of the complete spectrum of CGG-repeat mutation, with or without methylation assessment, and, as a result, have reduced our reliance on the labor- and time-intensive SB, which is the gold standard FXS diagnostic test. The newer and more robust triplet-primed PCR or TP-PCR assays allow the mapping of AGG interruptions and enable the predictive analysis of the risks of unstable CGG expansion during mother-to-child transmission. In this review, we have summarized the correlation between several molecular elements, including CGG-repeat size, methylation, mosaicism and skewed X-chromosome inactivation, and the extent of clinical involvement in patients with FMR1-related disorders, and reviewed key developments in PCR-based methodologies for the molecular diagnosis of FXS, FXTAS and FXPOI, and large-scale (CGG)n expansion screening in newborns, women of reproductive age and high-risk populations

Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders

Fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability and autism. Molecular diagnostic testing of FXS and related disorders (fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS)) relies on a combination of polymerase chain reaction (PCR) and Southern blot (SB) for the fragile X mental retardation 1 (FMR1) CGG-repeat expansion and methylation analyses. Recent advancements in PCR-based technologies have enabled the characterization of the complete spectrum of CGG-repeat mutation, with or without methylation assessment, and, as a result, have reduced our reliance on the labor- and time-intensive SB, which is the gold standard FXS diagnostic test. The newer and more robust triplet-primed PCR or TP-PCR assays allow the mapping of AGG interruptions and enable the predictive analysis of the risks of unstable CGG expansion during mother-to-child transmission. In this review, we have summarized the correlation between several molecular elements, including CGG-repeat size, methylation, mosaicism and skewed X-chromosome inactivation, and the extent of clinical involvement in patients with FMR1-related disorders, and reviewed key developments in PCR-based methodologies for the molecular diagnosis of FXS, FXTAS and FXPOI, and large-scale (CGG)n expansion screening in newborns, women of reproductive age and high-risk populations

Straglr: discovering and genotyping tandem repeat expansions using whole genome long-read sequences

Tandem repeat (TR) expansion is the underlying cause of over 40 neurological disorders. Long-read sequencing offers an exciting avenue over conventional technologies for detecting TR expansions. Here, we present Straglr, a robust software tool for both targeted genotyping and novel expansion detection from long-read alignments. We benchmark Straglr using various simulations, targeted genotyping data of cell lines carrying expansions of known diseases, and whole genome sequencing data with chromosome-scale assembly. Our results suggest that Straglr may be useful for investigating disease-associated TR expansions using long-read sequencing.Medicine, Faculty ofNon UBCMedical Genetics, Department ofReviewedFacult

High-Throughput Methylation-Specific Triplet-Primed PCR and Melting Curve Analysis for Selective and Reliable Identification of Actionable FMR1 Genotypes

10.1016/j.jmoldx.2021.11.007JOURNAL OF MOLECULAR DIAGNOSTICS243241-25

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome

10.1017/erm.2017.10EXPERT REVIEWS IN MOLECULAR MEDICINE1

Defining the Performance Parameters of a Rapid Screening Tool for FMR1 CGG-Repeat Expansions Based on Direct Triplet-Primed PCR and Melt Curve Analysis

10.1016/j.jmoldx.2016.05.002JOURNAL OF MOLECULAR DIAGNOSTICS185719-73

Electropherograms of samples 1–8 obtained from 3’ direct triplet primed PCR followed by capillary electrophoresis.

<p>(A)- (H): samples 1 to 8. Peaks in the eletropherogram indicate the number of CGG repeats in each individual.</p

Agarose gel electrophoresis of methylation specific PCR.

<p>(A), (B) and (C): samples 1 to 8. (D), (E) and (F): samples 9 to19. Top panel, non methylated PCR (Non Met PCR). Middle panel, methylated PCR (Met PCR). Bottom panel, methylated triplet primed PCR (mTP-PCR). L: l kb DNA molecular weight marker (Promega), N: Negative control (without genomic DNA).</p