title: Molecular diagnosis of fragile X syndrome using methylation sensitive techniques in a cohort of patients with intellectual disability.
abstract: BACKGROUND
  Fragile X syndrome, the most common form of inherited intellectual disability, is caused by expansion of CGG trinucleotide repeat at the 5' untranslated region of the FMR1 gene at Xq27. In affected individuals, the CGG repeat expansion leads to hypermethylation and the gene is transcriptionally inactive. Our aim was to identify fragile X syndrome among children with intellectual disability in Saudi Arabia.
  
  
  METHODS
  The study included 63 patients (53 males, 10 females) presented with intellectual disability, 29 normal subjects, and 23 other family members. DNA samples from six patients previously diagnosed with fragile X syndrome by Southern blot technique were used as positive controls. The method was based on bisulfite treatment of DNA followed by two different techniques. The first technique applied polymerase chain reaction amplification using one set of primers specific for amplifying methylated CpG dinucleotide region; another set designed to amplify the unmethylated CGG repeats. The second technique used the methylation-specific melting curve analysis for detection of methylation status of the FMR1 promoter region.
  
  
  RESULTS
  Molecular testing using methylation sensitive polymerase chain reaction had shown amplified products in all normal subjects using unmethylated but not methylated primers indicating normal alleles, whereas amplified products were obtained using methylated polymerase chain reaction primers in fragile X syndrome-positive samples and in 9 of 53 males, indicating affected individuals. Molecular testing using melting curve analysis has shown a single low melting peak in all normal males and in (44/53) patients indicating unmethylated FMR1 gene, whereas high melting peak indicating methylated gene was observed in the fragile X syndrome-positive samples and in 9 of 53 patients. We found 100% concordance between results of both techniques and the results of Southern blot analysis. Three samples have shown both methylated and unmethylated alleles, indicating possible mosaicism. No female patients or carriers could be detected by both techniques.
  
  
  CONCLUSIONS
  The technique can be applied for the rapid screening for fragile X syndrome among patients with intellectual disability. The impact of mosaicism on clinical severity needs further investigation.
authors.affiliation: Faculty of Medical Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
authors.forename: Adeel G
authors.initials: AG
authors.lastname: Chaudhary
authors.affiliation: Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia. Electronic address: irhussein@gmail.com.
authors.forename: Ibtessam R
authors.initials: IR
authors.lastname: Hussein
authors.affiliation: Faculty of Medical Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
authors.forename: Adel
authors.initials: A
authors.lastname: Abuzenadah
authors.affiliation: Faculty of Medical Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
authors.forename: Mamdouh
authors.initials: M
authors.lastname: Gari
authors.affiliation: Pediatric Hospital, Ministry of Health, Al Taif, Kingdom of Saudi Arabia.
authors.forename: Randa
authors.initials: R
authors.lastname: Bassiouni
authors.affiliation: King Fahd Hospital, Jeddah, Kingdom of Saudi Arabia.
authors.forename: Samira
authors.initials: S
authors.lastname: Sogaty
authors.affiliation: Faculty of Science, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
authors.forename: Sahira
authors.initials: S
authors.lastname: Lary
authors.affiliation: Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
authors.forename: Maha
authors.initials: M
authors.lastname: Al-Quaiti
authors.affiliation: King Abdulaziz Medical City for National Guard Health Affairs, and King Saud bin Abdulaziz University for Health Sciences, Riyadh, Kingdom of Saudi Arabia.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Al Balwi
authors.affiliation: Faculty of Medical Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Al Qahtani
mesh_terms: D000293:Adolescent; D000328:Adult; D015139:Blotting, Southern; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D004252:DNA Mutational Analysis / Q000379:methods; D005190:Family; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D007223:Infant; D008607:Intellectual Disability / Q000235:genetics*; D008297:Male; D008745:Methylation; D025202:Molecular Diagnostic Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D018911:Trinucleotide Repeats; D055815:Young Adult
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human
keywords: Fragile X; intellectual disability; melting curve analysis; methylation sensitive PCR; molecular diagnosis