A Rapid Polymerase Chain Reaction-Based Screening Method for Identification of All Expanded Alleles of the Fragile X (FMR1) Gene in Newborn and High-Risk Populations

Abstract

Fragile X syndrome , the most common inherited cause of intellectual impairment and the most common single gene associated with autism , generally occurs for fragile X mental retardation 1 (FMR1) alleles that exceed 200 CGG repeats (full-mutation range). Currently, there are no unbiased estimates of the number of full-mutation FMR1 alleles in the general population; a major obstacle is the lack of an effective screening tool for expanded FMR1 alleles in large populations. We have developed a rapid polymerase chain reaction (PCR)-based screening tool for expanded FMR1 alleles. The method utilizes a chimeric PCR primer that targets randomly within the expanded CGG region , such that the presence of a broad distribution of PCR products represents a positive result for an expanded allele. The method is applicable for screening both males and females and for allele sizes throughout the premutation (55 to 200 CGG repeats) and full-mutation ranges. Furthermore , the method is capable of rapid detection of expanded alleles using as little as 1% of the DNA from a single dried blood spot. The methodology presented in this work is suitable for screening large populations of newborn or those at high risk (eg , autism , premature ovarian failure , ataxia , dementia) for expanded FMR1 alleles. The test described herein costs less than $5 per sample for materials; with suitable scale-up and automation , the cost should approach $1 per sample. (J Mol

Screening for Expanded Alleles of the FMR1 Gene in Blood Spots from Newborn Males in a Spanish Population

“…Samples that did not yield a band after the first round PCR with primers c and f were subjected to a secondary CGG-primer-based PCR screening as previously described. 18 Figure 2 shows PCR products obtained from secondary screening of blood spots using the chimeric CGG-targeted primer for the detection of large CGG repeat expansions run on a 2% agarose gel. An extensive smear is produced with the chimeric primer when an expanded allele is present, as shown in lanes 1 and 4 for the two full mutation samples, while no smear is visible in the presence of a normal allele as shown in lanes 2 and 3.…”

“…Blood spots that underwent the CGG primer-based PCR screening were washed two times for 15 minutes in 1 ml of ddH2O and used immediately in a PCR reaction. 18 PCR products were run on a 2% agarose gel. Isolation of DNA from blood spots was performed on the two full mutation samples to rule out the possibility that the two samples were indeed large premutation alleles (see Results).…”

“…However, with the recent development of such a test, 18 screening of blood spots to detect all expanded alleles is now feasible, which in turn permits large-scale screening of newborn populations.…”

Caracterización citogenético molecular de enfermedades genéticas en el Hospital Base de Puerto Montt

“…Brevemente, los estudios de FISH de secuencia única para cromosomas 7,15, 22, X e Y, así como de regiones subteloméricas se realizaron en metafases obtenidas de cultivos de linfocitos, utilizando sondas fl uorescentes específi cas (Vysis) y visualizadas al microscopio de fl uorescencia (Olympus). Para los tests de metilación y PCRs se aisló ADN del paciente utilizando un kit de extracción (Wizard® Genomic DNA Purifi cation, Promega) y se procedió a realizar los estudios respectivos según protocolos establecidos y adaptados en el laboratorio del INTA 25 ; para el test de metilación se utilizó el protocolo establecido por Kosaki y cols, 1997 26 y para el diagnóstico del SXF los protocolos de Saluto y cols 27 y Tassone y cols 28 .…”

“It gives them more options”: preferences for preconception genetic carrier screening for fragile X syndrome in primary healthcare

“…As such, FXS carrier screening, either stand-alone or as part of expanded screening, provides a useful model through which to consider carrier screening approaches because of the complexity and duality of risk information provided (Henneman et al 2016). As outlined by Hill et al (2010), arguments for carrier screening for FXS include a high prevalence (which may be up to 1 in 2500 in males and females (Hagerman 2008)) and high carrier frequency (recently reported to be 1 in 179 estimated for North American populations (Hantash et al 2011)), the prevalence of premutation alleles estimated as 1:209 in females and 1:430 in males (Tassone et al 2012), and an accurate testing method amenable to screening populations (Tassone et al 2008) which identifies an expanded CGG repeat in the FMR1 gene (the cause of FXS in over 99 % of cases). In addition, the condition is well understood and defined; there are a range of significant phenotypic effects associated with the full mutation (FM ≥200 hypermethylated CGG repeats) including intellectual disability and cognitive, behavioral, and medical problems, which have a substantial impact on the affected individual and their family (Hagerman and Hagerman 2002).…”