title: Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis.
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.
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia; Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia; Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile;
authors.forename: Solange M
authors.initials: SM
authors.lastname: Aliaga
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia; Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia;
authors.forename: Howard R
authors.initials: HR
authors.lastname: Slater
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia;
authors.forename: David
authors.initials: D
authors.lastname: Francis
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia;
authors.forename: Desiree
authors.initials: D
authors.lastname: Du Sart
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia;
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia; Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia;
authors.forename: David J
authors.initials: DJ
authors.lastname: Amor
authors.affiliation: Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile.
authors.forename: Angelica M
authors.initials: AM
authors.lastname: Alliende
authors.affiliation: Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile; Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile.
authors.forename: Lorena
authors.initials: L
authors.lastname: Santa Maria
authors.affiliation: Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile; Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile.
authors.forename: Víctor
authors.initials: V
authors.lastname: Faundes
authors.affiliation: Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile; Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile.
authors.forename: Paulina
authors.initials: P
authors.lastname: Morales
authors.affiliation: Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile.
authors.forename: Cesar
authors.initials: C
authors.lastname: Trigo
authors.affiliation: Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile.
authors.forename: Isabel
authors.initials: I
authors.lastname: Salas
authors.affiliation: Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile; Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile.
authors.forename: Bianca
authors.initials: B
authors.lastname: Curotto
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia; david.godler@mcri.edu.au.
authors.forename: David E
authors.initials: DE
authors.lastname: Godler
mesh_terms: D000293:Adolescent; D000328:Adult; D000483:Alleles; D015139:Blotting, Southern; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D018899:CpG Islands; D019175:DNA Methylation; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics / Q000378:metabolism; D005600:Fragile X Syndrome / Q000235:genetics*; D005821:Genetic Techniques; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D009030:Mosaicism; D016133:Polymerase Chain Reaction / Q000379:methods; D055815:Young Adult
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human