Fragile X Syndrome Mutations Found With Comprehensive Testing Method

Fragile X syndrome (FXS), the most common form of inherited intellectual disability and monogenic cause of autism spectrum disorders, is mainly caused by the expansion of CGG trinucleotide repeats in the 5′ untranslated region of FMR1 gene.

Individuals with a premutation allele are at risk for fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X-associated neuropsychiatric disorders. The premutation allele can expand to a full mutation allele during female germline transmission, thus being transmitted to the offspring with FXS.

Medical Geneticists at the Central South University (Changsha, China) and their associates retrospectively enrolled a total of 62 genomic DNA samples from 21 families with a history of FXS from the diagnostic laboratory in Hunan Jiahui Genetics Hospital, including 57 samples from peripheral blood and five from chorionic villus sampling. All 21 families had members with FMR1 variants identified by in-house triplet repeat-primed – PCR (TP–PCR) assay, Southern Blot Analysis (SBA), Sanger sequencing, and Gap-PCR.

TP–PCR and gene-specific PCR (GS-PCR) were performed using AmplideX PCR/CE FMR1 Reagents from Asuragen (Austin, TX, USA) and analyzed on 3130 ABI Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). PCR and Sanger sequencing of FMR1 exons and exon-intron boundaries were performed to analyze rare intragenic variants. Gap-PCR and Sanger sequencing were performed to determine the breakpoints of microdeletions. Comprehensive analysis of FXS (CAFXS) assay included two reactions for comprehensive analysis of FMR1 variants. Southern Blot Analysis of digested genomic DNA was also performed.

The investigators reported that CAFXS accurately detected the number of CGG repeats in the range of 93 to at least 940 with mass fraction of 0.5% to 1% in the background of normal alleles, which was 2–4-fold analytically more sensitive than TP–PCR. All categories of mutations detected by control methods, including full mutations in 30 samples, were identified by CAFXS for all 62 clinical samples. CAFXS accurately determined AGG interruptions in all 133 alleles identified, even in mosaic alleles. CAFXS successfully identified two rare intragenic variants including the c.879A > C variant in exon 9 and a 697-bp microdeletion flanking upstream of CGG repeats, which disrupted primer annealing in TP–PCR assay. In addition, CAFXS directly determined the breakpoints of a 237.1-kb deletion and a 774.0-kb deletion encompassing the entire FMR1 gene in two samples.

The authors concluded that long-read sequencing-based CAFXS represents a comprehensive assay for identifying FMR1 CGG expansions, AGG interruptions, rare intragenic variants, and large gene deletions, which greatly improves the genetic screening and diagnosis for FXS. The study was published on September 29, 2022 in the journal Clinical Chemistry.

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