Solange M Aliaga1, Howard R Slater2, David Francis3, Desiree Du Sart3, Xin Li3, David J Amor2, Angelica M Alliende4, Lorena Santa Maria5, Víctor Faundes5, Paulina Morales5, Cesar Trigo4, Isabel Salas4, Bianca Curotto5, David E Godler6. 1. 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; 2. 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; 3. Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia; 4. Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile. 5. 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. 6. 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.
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.
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: David J Amor; David E Godler; Emma K Baker; Marta Arpone; Solange M Aliaga; Lesley Bretherton; Claudine M Kraan; Minh Bui; Howard R Slater; Ling Ling; David Francis; Matthew F Hunter; Justine Elliott; Carolyn Rogers; Michael Field; Jonathan Cohen; Kim Cornish; Lorena Santa Maria; Victor Faundes; Bianca Curotto; Paulina Morales; Cesar Trigo; Isabel Salas; Angelica M Alliende Journal: Mol Autism Date: 2019-05-03 Impact factor: 7.509
Authors: Alison Pandelache; Emma K Baker; Solange M Aliaga; Marta Arpone; Robin Forbes; Zornitza Stark; David Francis; David E Godler Journal: Genes (Basel) Date: 2019-04-05 Impact factor: 4.096
Authors: Michael Field; Tracy Dudding-Byth; Marta Arpone; Emma K Baker; Solange M Aliaga; Carolyn Rogers; Chriselle Hickerton; David Francis; Dean G Phelan; Elizabeth E Palmer; David J Amor; Howard Slater; Lesley Bretherton; Ling Ling; David E Godler Journal: Int J Mol Sci Date: 2019-08-11 Impact factor: 5.923
Authors: Marta Arpone; Emma K Baker; Lesley Bretherton; Minh Bui; Xin Li; Simon Whitaker; Cheryl Dissanayake; Jonathan Cohen; Chriselle Hickerton; Carolyn Rogers; Mike Field; Justine Elliott; Solange M Aliaga; Ling Ling; David Francis; Stephen J C Hearps; Matthew F Hunter; David J Amor; David E Godler Journal: Sci Rep Date: 2018-02-26 Impact factor: 4.379
Authors: Charles H Hensel; Rena J Vanzo; Megan M Martin; Ling Ling; Solange M Aliaga; Minh Bui; David I Francis; Hope Twede; Michael H Field; Jonathon W Morison; David J Amor; David E Godler Journal: Sci Rep Date: 2019-10-25 Impact factor: 4.379