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Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis

2015; American Association for Clinical Chemistry; Volume: 62; Issue: 2 Linguagem: Inglês

10.1373/clinchem.2015.244681

1530-8561

Solange Aliaga, Howard R. Slater, David Francis, Desirée du Sart, Xin Li, David J. Amor, Angelica M. Alliende, Lorena Santa María, Víctor Faúndes, Paulina Morales, César Trigo, Isabel Salas, Bianca Curotto, David E. Godler,

Epigenetics and DNA Methylation

Abstract BACKGROUND FMR1 full mutations (FMs) (CGG expansion >200) in males mosaic for a normal (<45 CGG) or gray-zone (GZ) (45–54 CGG) allele can be missed with the standard 2-step fragile X syndrome (FXS) testing protocols, largely because the first-line PCR tests showing a normal or GZ allele are not reflexed to the second-line test that can detect FM. METHODS We used methylation-specific quantitative melt analysis (MS-QMA) to determine the prevalence of cryptic FM alleles in 2 independent cohorts of male patients (994 from Chile and 2392 from Australia) referred for FXS testing from 2006 to 2013. All MS-QMA–positive cases were retested with commercial triplet primed PCR, methylation-sensitive Southern blot, and a methylation-specific EpiTYPER-based test. RESULTS All 38 FMs detected with the standard 2-step protocol were detected with MS-QMA. However, MS-QMA identified methylation mosaicism in an additional 15% and 11% of patients in the Chilean and Australian cohorts, respectively, suggesting the presence of a cryptic FM. Of these additional patients, 57% were confirmed to carry cryptic expanded alleles in blood, buccal mucosa, or saliva samples. Further confirmation was provided by identifying premutation (CGG 55–199) alleles in mothers of probands with methylation-sensitive Southern blot. Neurocognitive assessments showed that low-level mosaicism for cryptic FM alleles was associated with cognitive impairment or autism. CONCLUSIONS A substantial number of mosaic FM males who have cognitive impairment or autism are not diagnosed with the currently recommended 2-step testing protocol and can be identified with MS-QMA as a first-line test.