Both cis and trans-acting genetic factors drive somatic instability in female carriers of the FMR1 premutation

Abstract

The fragile X mental retardation (FMR1) gene contains an expansion-prone CGG repeat within its 5′ UTR. Alleles with 55–200 repeats are known as premutation (PM) alleles and confer risk for one or more of the FMR1 premutation (PM) disorders that include Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), Fragile X-associated Primary Ovarian Insufficiency (FXPOI), and Fragile X-Associated Neuropsychiatric Disorders (FXAND). PM alleles expand on intergenerational transmission, with the children of PM mothers being at risk of inheriting alleles with > 200 CGG repeats (full mutation FM) alleles) and thus developing Fragile X Syndrome (FXS). PM alleles can be somatically unstable. This can lead to individuals being mosaic for multiple size alleles. Here, we describe a detailed evaluation of somatic mosaicism in a large cohort of female PM carriers and show that 94% display some evidence of somatic instability with the presence of a series of expanded alleles that differ from the next allele by a single repeat unit. Using two different metrics for instability that we have developed, we show that, as with intergenerational instability, there is a direct relationship between the extent of somatic expansion and the number of CGG repeats in the originally inherited allele and an inverse relationship with the number of AGG interruptions. Expansions are progressive as evidenced by a positive correlation with age and by examination of blood samples from the same individual taken at different time points. Our data also suggests the existence of other genetic or environmental factors that affect the extent of somatic expansion. Importantly, the analysis of candidate single nucleotide polymorphisms (SNPs) suggests that two DNA repair factors, FAN1 and MSH3, may be modifiers of somatic expansion risk in the PM population as observed in other repeat expansion disorders.

Clinical implications of somatic allele expansion in female FMR1 premutation carriers

“…Instability of the FMR1 premutation allele has recently been described for the first time by our group on a large sample of female PM 18 . Specifically, we characterized the presence of a series of unstable expanded alleles in ~ 94% of peripheral blood samples differing from the major premutation allele by one or more repeat units.…”

“…The expanded peak with the highest relative fluorescence units (RFU) value was selected as Peak 2. Expansion was calculated using the formula (Peak 2–Peak 1), more details can be found in Hwang et al 18 . Total RNA was isolated from 2.5 ml of peripheral blood collected in PAXgene (Qiagen, Valencia, CA, United States) and quantified using the Agilent 2100 Bioanalyzer system.…”

“…Specific measures analyzed in the FXPOI group was the age at menopause and the presence of FXPOI while that in the FXTAS group was the presence and the stage of FXTAS. Expansion was measured as described in our previous paper 18 but in essence, instability was represented by ∆Rpts, which, in a repeat profile, represents the difference in the number of repeats between the modal expanded allele (Peak 2) and modal stable allele (Peak 1). Spearman test of correlation were used to examine CGG repeat length, FMR1 mRNA expression, and expansion (measured as peak 2—peak 1) against IQ measures.…”

Variation of FMRP Expression in Peripheral Blood Mononuclear Cells from Individuals with Fragile X Syndrome

“…While expansions and, to a lesser degree, contractions, occur in any allele class, PM alleles are particularly meiotically unstable, often expanding into FM alleles in a single generation [ 40 , 42 ]. More recently, it has been shown that somatic mosaicism is also common in PM alleles and that genes involved in DNA repair may play a role in somatic expansion risk, as reported in other repeat-expansion disorders [ 43 ].…”

Mismatch repair is a double-edged sword in the battle against microsatellite instability

“…In contrast, studies in RED patient cohorts using genome-wide association (GWA) or the testing of candidate MMR gene polymorphisms suggest that functional MMR components are required for some, if not all, STR expansions that cause the REDs (Refs 69 – 74 ). This is consistent with evidence from mouse and human cell models of a number of these disorders that shows a requirement for MutS β and MutL γ (reviewed in Ref.…”