Dataset Information

A Single Common Assay for Robust and Rapid Fragile X Mental Retardation Syndrome Screening From Dried Blood Spots.

ABSTRACT: Background: FMR1 CGG trinucleotide repeat hyper-expansions are observed in 99% of individuals with fragile X mental retardation syndrome (FXS). We evaluated the reliability of a rapid single-step gender-neutral molecular screen for FXS when performed on DNA isolated from dried blood spots. Methods: DNA was extracted from dried blood spots of 151 individuals with intellectual disability or autism spectrum disorder, whose FMR1 repeat genotypes are known. Dried blood spots were blinded prior to DNA extraction and analysis by triplet primed PCR (TP-PCR) and melt curve analysis (MCA). All expansion-positive and representative expansion-negative samples were also genotyped by fluorescent TP-PCR and capillary electrophoresis (CE) to confirm repeat expansion status. Results: Three males and 12 females were classified as expanded by TP-PCR MCA, and were subsequently sized by fluorescent TP-PCR CE. Two males and four females carried premutations, while one male and eight females carried full mutations. All 19 non-expanded samples that were sized were confirmed as carrying only normal alleles. Replicate analysis of representative expansion-positive samples yielded reproducible melt peak profiles. TP-PCR MCA classifications were completely concordant with FMR1 CGG repeat genotypes. Conclusion: TP-PCR MCA of dried blood spot DNA accurately and reliably identifies presence/absence of FMR1 CGG repeat expansions in both genders simultaneously. This strategy may be suitable for rapid high-throughput first-tier screening for fragile X syndrome.

SUBMITTER: Tan VJ

PROVIDER: S-EPMC6277581 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A Single Common Assay for Robust and Rapid Fragile X Mental Retardation Syndrome Screening From Dried Blood Spots.

Tan Vivienne J VJ Lian Mulias M Faradz Sultana M H SMH Winarni Tri I TI Chong Samuel S SS

Frontiers in genetics 20181127

Background: FMR1 CGG trinucleotide repeat hyper-expansions are observed in 99% of individuals with fragile X mental retardation syndrome (FXS). We evaluated the reliability of a rapid single-step gender-neutral molecular screen for FXS when performed on DNA isolated from dried blood spots. Methods: DNA was extracted from dried blood spots of 151 individuals with intellectual disability or autism spectrum disorder, whose FMR1 repeat genotypes are known. Dried blood spo ...[more]

PMID: 30538724

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Project description:Background: Primary immunodeficiency disorders (PIDD) comprise a group of life-threatening congenital diseases characterized by absent or impaired immune responses. Despite the fact that effective, curative treatments are available with optimal clinical outcomes when diagnosed early, newborn screening does not exist for the majority of these diseases due to the lack of detectable, specific biomarkers or validated methods for population-based screening. Peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) is a sensitive proteomic assay, involving antibody-mediated peptide capture, that allows for concurrent quantification of multiple analytes. This assay has promise for use in potential newborn screening of PIDDs that lead to diminished or absent target proteins in the majority of cases. Objective: To determine and evaluate if a multiplex assay based on immuno-SRM is able to reliably and precisely distinguish affected patients with X-linked agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), and CD3?-associated severe combined immunodeficiency (SCID) from one another and from unaffected normal control dried blood spot (DBS) samples. Methods: We performed a blinded, multiplexed analysis of proteolytically-generated peptides from WASp, BTK, and CD3? (for WAS, XLA, and SCID, respectively) in DBS samples from 42 PIDD patients, 40 normal adult controls, and 62 normal newborns. The peptide ATPase copper transporting protein (ATP7B) 1056 was simultaneously monitored for quality assurance purposes. Results: The immuno-SRM assays reliably quantified the target peptides in DBS and accurately distinguished affected patients from normal controls. Analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (WASp and BTK: p = 0.0001, SCID: p = 0.05). Intra and inter-assay precision ranged from 11 to 22% and 11 to 43% respectively; linearity (1.39-2000 fmol peptide), and stability (? 0.09% difference in 72 h) showed high precision for the multiplexed assay. Inter-laboratory assay comparison showed high concordance for measured peptide concentrations, with R2 linearity ? 0.97 for the WASp 274, CD3? 197, BTK 407, and ATP7B 1056 peptides. Conclusion: Immuno-SRM-based quantification of proteotypic peptides from WASp, BTK, and CD3? in DBS distinguishes relevant PIDD cases from one another and from controls, raising the possibility of employing this approach for large-scale multiplexed newborn screening of selective PIDDs.

Dataset Information

A Single Common Assay for Robust and Rapid Fragile X Mental Retardation Syndrome Screening From Dried Blood Spots.

Publications

A Single Common Assay for Robust and Rapid Fragile X Mental Retardation Syndrome Screening From Dried Blood Spots.

Similar Datasets

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