Consensus Characterization of 16 FMR1 Reference Materials: A Consortium Study

Abstract

Fragile X syndrome , which is caused by expansion of a (CGG) n repeat in the FMR1 gene , occurs in approximately 1:3500 males and causes mental retardation/ behavioral problems. Smaller (CGG) n repeat expansions in FMR1, premutations , are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG) n repeat , the size limitations of conventional PCR , and a lack of reference materials available for test development/validation and routine quality Fragile X syndrome (FXS) is the most common inherited cause of mental retardation, with an incidence in males of approximately 1 in 3500 (for a recent review, see http:// genetests.org). Clinical features in males include mental retardation, specific physical characteristics (enlarged testes, large ears, and long face), and behavioral abnormalities, sometimes including autism spectrum disorder. Affected females, with an incidence of approximately 1 in 8000, have mild mental retardation. Our knowledge of the spectrum of phenotypes associated with expansion of the FMR1 gene now also includes premature ovarian failure 1 and fragile X (FX)-associated tremor/ataxia syndrome.2-5 Thus, genetic testing for FX mutations is important at all life stages, prenatally to adulthood.

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

“…This absolute quantification approach was free from the need for a reference calibration standard, such as the internal ROX standard. The accuracy of triplet repeat quantification using this approach was well correlated to the results of Amos Wilson et al 13 using published fragile X consensus materials (Table 1). For example, determination of the (CGG) n repeat length exactly matched 12 of 14 FMR1 alleles whose consensus length were previously determined.…”

“…Thus, the RP peak profile can theoretically provide very accurate (CGG) n repeat quantification. In our study, (CGG) n repeat lengths for 12 of 14 alleles previously characterized in an interlaboratory study of the Fragile Xperts Working Group 13 were identical to those determined using absolute quantification after CGG RP PCR. Discrepancies in the repeat number were 1 or 3 triplet repeats.…”

“…For example, determination of the (CGG) n repeat length exactly matched 12 of 14 FMR1 alleles whose consensus length were previously determined. 13 The remaining two alleles differed by 1 CGG (74 versus 73 CGG) or 3 CGG (83 versus 80 CGG) repeats.…”

Screening for CGG Repeat Expansion in the FMR1 Gene by Melting Curve Analysis of Combined 5′ and 3′ Direct Triplet-Primed PCRs

“…The assay was further validated with 23 additional DNA samples, 15 of which were from a panel of previously characterized fragile X mutation reference DNA samples, with critical diagnostic cutoffs (24 ). A final blinded validation analysis was performed with 44 archived whole bloodderived clinical samples enriched for PM and FM alleles.…”

“…These samples consist of 6 NL males, 1 PM male, 14 FM males, 5 NL females, 9 PM females, and 9 FM females. All samples were previously characterized by PCR and/or Southern blot analysis (24,25 ).…”