Molecular testing for Fragile X Syndrome: Lessons learned from 119,232 tests performed in a clinical laboratory

Strom, Charles M.; Crossley, Beryl; Redman, Joy B.; Buller, Arlene; Quan, Franklin; Peng, Mei; McGinnis, Matthew; Fenwick, Raymond G.; Sun, Weimin

doi:10.1097/gim.0b013e31802d833c

Cited by 55 publications

(42 citation statements)

References 18 publications

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“…As a result, the capabilities of the RP PCR may reduce the burden of Southern blot analysis to only those samples that require methylation information, which are typically Յ2% of the total samples processed by large clinical reference laboratories. 23 Moreover, the PCR reagents described both here and previously 8 may support PCRbased methylation assessments of FMR1 alleles using methylation-sensitive restriction enzymes. As a result, the RP PCR technology, and the corresponding gene-specific PCR technology, 8 represents an important step toward a PCR-only workflow for FMR1 analysis.…”

Section: Discussionmentioning

confidence: 99%

An Information-Rich CGG Repeat Primed PCR That Detects the Full Range of Fragile X Expanded Alleles and Minimizes the Need for Southern Blot Analysis

Chen

Hadd

Sah

et al. 2010

The Journal of Molecular Diagnostics

165

199

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(CGG) n repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel , single-tube CGG repeat primed FMR1 PCR technology was designed with two genespecific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG) n repeat. This PCR was evaluated with 171 unique DNA samples , including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis , including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore , a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles , was resolved with 100% accuracy. Last , AGG interrupter sequences, which may influence the risk of (CGG) n expansion in the children of some carriers , were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result , this PCR provides robust detection of expanded alleles and resolves allele zygosity , thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods. Expansion of cytosine-guanine-guanine (CGG) triplet repeats in the 5Ј-untranslated region of the fragile X mental retardation 1 (FMR1, NM_002024.4) gene is associated with several disorders, including fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. [1][2][3][4] Patients with the FMR1 full mutation (Ͼ200 CGG repeats) may be affected by a range of neurological, psychiatric, or emotional challenges, including mental retardation and/or autism.5 Deficits in development and particularly in attention and social communication have also been noted for many children with the FMR1 premutation. Moreover, premutation carriers (55 to 200 CGG repeats) are known to be at risk for fragile X-associated primary ovarian insufficiency and fragile X-associated tremor/ataxia syndrome, and some of these individuals may present additional complications, such as hypothyroidism and fibromyalgia.6 As a result, FMR1 disorders are linked to a range of clinical conditions, necessitating testing patients at different times during their life span. 7Fragile X syndrome molecular diagnosis is usually based on quantification of the (CGG) n repeat elements and the assessment of the methylation state of expanded alleles.5 Although PCR is the preferred approach to determine the (CGG) n repeat length of FMR1 alleles, typically only alleles with less than 100 to 150 CGG have

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Section: Discussionmentioning

confidence: 99%

An Information-Rich CGG Repeat Primed PCR That Detects the Full Range of Fragile X Expanded Alleles and Minimizes the Need for Southern Blot Analysis

Chen

Hadd

Sah

et al. 2010

The Journal of Molecular Diagnostics

165

199

View full text Add to dashboard Cite

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“…42 In the largest US study to date (of over 119,000 tested), the overall carrier frequency among US females was 1.3% (0.61% for full mutation and 1.7% for premutation). 43 A recent large-scale study of 21,411 anonymous Canadian females (mothers of newborns) identified 1 in 549 as carriers. 34 Previous screens for the prevalence of premutations (with 55-101 repeats) in French-Canadian women estimated the carrier frequency to be 1 in 259.…”

Section: Fx 282: Premutationsmentioning

confidence: 99%

ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics

Monaghan

Lyon

Spector

2013

Genetics in Medicine

129

137

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“…Indeed, only B1.4% of patients clinically tested for the full mutation are positive. 10 Partly, this is due to the nonspecific and variable phenotype of FXS and that testing for FXS is among the primary tests ordered for any child not reaching developmental milestones, therefore a low positive test rate is expected. Yet missense FMR1 mutations must exist in the population, perhaps leading to phenotypes that are not usually brought to medical attention, such as children with learning disabilities or who struggle in regular classrooms but not considered intellectually impaired.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, the actual diagnostic yield is only 1-2%. 10 Whereas it is clear that CGG-repeat expansion is the most frequent cause of FXS, more conventional mutations, particularly FMR1 deletions, have also been reported in FXS. 11 Deletions are typically found as an anomaly of the CGG-repeat test or by microarray analysis, and sequencing of FMR1 is not frequently carried out.…”

Section: Introductionmentioning

confidence: 99%

Fragile X syndrome due to a missense mutation

et al. 2014

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Fragile X syndrome is a common inherited form of intellectual disability and autism spectrum disorder. Most patients exhibit a massive CGG-repeat expansion mutation in the FMR1 gene that silences the locus. In over two decades since the discovery of FMR1, only a single missense mutation (p.(Ile304Asn)) has been reported as causing fragile X syndrome. Here we describe a 16-year-old male presenting with fragile X syndrome but without the repeat expansion mutation. Rather, we find a missense mutation, c.797G4A, that replaces glycine 266 with glutamic acid (p. (Gly266Glu)). The Gly266Glu FMR protein abolished many functional properties of the protein. This patient highlights the diagnostic utility of FMR1 sequencing.

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Molecular testing for Fragile X Syndrome: Lessons learned from 119,232 tests performed in a clinical laboratory

Cited by 55 publications

References 18 publications

An Information-Rich CGG Repeat Primed PCR That Detects the Full Range of Fragile X Expanded Alleles and Minimizes the Need for Southern Blot Analysis

An Information-Rich CGG Repeat Primed PCR That Detects the Full Range of Fragile X Expanded Alleles and Minimizes the Need for Southern Blot Analysis

ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics

Fragile X syndrome due to a missense mutation

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