title: Development and validation of a multiplex-PCR assay for X-linked intellectual disability.
abstract: BACKGROUND
  X-linked intellectual disability is a common cause of inherited cognitive deficit affecting mostly males. There are several genetic causes implicated in this condition, which has hampered the establishment of an accurate diagnosis. We developed a multiplex-PCR assay for the mutational hotspot regions of the FMR1, AFF2 and ARX genes.
  
  
  METHODS
  The multiplex-PCR was validated in a cohort of 100 males selected to include known alleles for the FMR1 repetitive region: five full mutations (250-650 CGGs), ten premutations (70-165 CGGs) and eighty-five in the normal range (19-42 CGGs). Sequencing or Southern blotting was used to confirm the results, depending on the allele class. In this cohort, with the exception of one sample showing an AFF2 intermediate-sized allele, all other samples were normal (8-34 CCGs). No ARX variant was found besides the c.429_452dup. The validated assay was applied to 5000 samples (64.4% males and 35.6% females).
  
  
  RESULTS
  The normal-allelic range of both FMR1 and AFF2 genes as well as the nature of ARX variants identified was similar in both genders. The rate of homozygosity observed in female samples, 27.5% for FMR1 and 17.8% for AFF2 alleles, is comparable to that published by others. Two FMR1 premutations were identified, in a male (58 CGGs) and a female case [(CGG)(47)/(CGG)(61)], as well as several FMR1 or AFF2 intermediate-sized alleles. One AFF2 premutation (68 CCGs) and two putative full expansions were picked up in male subjects, which seems relevant considering the rarity of reported AFF2 mutations found in the absence of a family history.
  
  
  CONCLUSIONS
  We developed a robust multiplex-PCR that can be used to screen the mutational hotspot regions of FMR1, AFF2 and ARX genes. Moreover, this strategy led to the identification of variants in all three genes, representing not only an improvement in allele-sizing but also in achieving a differential diagnosis. Although the distinction between females who are truly homozygous and those with a second pre- or full mutation sized allele, as well as a definitive diagnosis, requires a specific downstream technique, the use of this multiplex-PCR for initial screening is a cost-effective approach which widens the scope of detection.
authors.affiliation: Centro de Genética Médica Dr, Jacinto Magalhães, CHP, Praça Pedro Nunes 88, 4099-028, Porto, Portugal. jorgpaula@gmail.com
authors.forename: Paula
authors.initials: P
authors.lastname: Jorge
authors.forename: Bárbara
authors.initials: B
authors.lastname: Oliveira
authors.forename: Isabel
authors.initials: I
authors.lastname: Marques
authors.forename: Rosário
authors.initials: R
authors.lastname: Santos
mesh_terms: D000483:Alleles; D015139:Blotting, Southern; D015331:Cohort Studies; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics; D005600:Fragile X Syndrome / Q000175:diagnosis / Q000235:genetics; D050172:Genes, X-Linked; D005820:Genetic Testing; D018398:Homeodomain Proteins / Q000235:genetics; D006720:Homozygote; D006801:Humans; D008607:Intellectual Disability / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D010641:Phenotype; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; C099930:AFF2 protein, human; C469264:ARX protein, human; C497683:FMR1 protein, human
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