An Information-Rich CGG Repeat Primed PCR That Detects the Full Range of Fragile X Expanded Alleles and Minimizes the Need for Southern Blot Analysis

Abstract

(CGG) n repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel , single-tube CGG repeat primed FMR1 PCR technology was designed with two genespecific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG) n repeat. This PCR was evaluated with 171 unique DNA samples , including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis , including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore , a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles , was resolved with 100% accuracy. Last , AGG interrupter sequences, which may influence the risk of (CGG) n expansion in the children of some carriers , were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result , this PCR provides robust detection of expanded alleles and resolves allele zygosity , thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods. Expansion of cytosine-guanine-guanine (CGG) triplet repeats in the 5Ј-untranslated region of the fragile X mental retardation 1 (FMR1, NM_002024.4) gene is associated with several disorders, including fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. [1][2][3][4] Patients with the FMR1 full mutation (Ͼ200 CGG repeats) may be affected by a range of neurological, psychiatric, or emotional challenges, including mental retardation and/or autism.5 Deficits in development and particularly in attention and social communication have also been noted for many children with the FMR1 premutation. Moreover, premutation carriers (55 to 200 CGG repeats) are known to be at risk for fragile X-associated primary ovarian insufficiency and fragile X-associated tremor/ataxia syndrome, and some of these individuals may present additional complications, such as hypothyroidism and fibromyalgia.6 As a result, FMR1 disorders are linked to a range of clinical conditions, necessitating testing patients at different times during their life span. 7Fragile X syndrome molecular diagnosis is usually based on quantification of the (CGG) n repeat elements and the assessment of the methylation state of expanded alleles.5 Although PCR is the preferred approach to determine the (CGG) n repeat length of FMR1 alleles, typically only alleles with less than 100 to 150 CGG have

Fragile X full mutation expansions are inhibited by one or more AGG interruptions in premutation carriers

“…Recent advances in polymerase chain reaction (PCR) technologies have enabled larger cohort studies and examination of AGG status in females. 9,12 These studies have affirmed the influence of AGG interruptions on instability and risk of full mutation expansion. 13,14 We previously examined 457 maternal and 81 paternal transmissions for alleles with 45-69 repeats.…”

“…All analyses were stratified by parental origin of the transmission. Full mutation alleles were further characterized using gene-specific PCR 12 and FlashGel electrophoresis with 1.2% precast agarose gels (Lonza, Basel, Switzerland). Full mutation allele sizes were estimated from a Quick-Load 2-Log DNA ladder (New England Biolabs, Ipswich, MA).…”

Identification of Expanded Alleles of theFMR1Gene Among High-Risk Population in Indonesia by Using Blood Spot Screening

“…The CE results were analyzed by using the ABI Peak Scanner software (Applied Biosystems). DNA samples from men that did not yield a band after the first round of PCR, or DNA samples from females that yielded only one normal band with primers c and f, were subjected to a secondary, chimeric-CGG-primer-based PCR screening, as previously described (Tassone et al, 2008;Chen et al, 2010). The PCR amplicons were subjected to visualization on an agarose gel (Tassone et al, 2008) or by (CE) (Chen et al, 2010).…”

Ethnic effect on FMR1 carrier rate and AGG repeat interruptions among Ashkenazi women

“…23 Two new PCR-based methods were introduced between 2009 and 2011, and they were used in cases indicated for Southern blot analysis as described earlier. The Abbott Fragile X PCR Kit (Abbott, Abbott Park, IL) 24 was used in 2009 and 2010, and the AmplideX FMR1 PCR kit (Asuragen, Austin, TX) 25 was used during 2011. In addition to the CCG repeat length, the PCR products generated with the CGG repeat primer used in the AmplideX kit reveals the number of AGG interspersions in a straightforward manner, as described in detail by Chen et al 25 We therefore used this additional information in the 830 cases that were analyzed by the AmplideX kit and recorded the number of AGG interruptions in addition to the number of CGG repeats in each case.…”

“…Similarly, a previous study detected a decreased diversity in the background haplotypes of the FMR1 gene among Ashkenazi Jews, with a higher incidence of a specific DXS548-FRAXAC1-FRAXAC2 haplotype (734+) in the Ashkenazi population. 18 Another study 25 did not find a founder effect in Ashkenazi patients with FXS but rather detected a high incidence of the 734+ haplotype in the Ashkenazi control group. 26 Importantly, this specific haplotype was shown to be associated with a decreased risk of CGG expansion among members of a nonJewish population.…”