The SALSA MS-MLPA
Probemix ME029 FMR1-AFF2 is a research use only (RUO)
assay for the detection of aberrant methylation of one or more sequences of the FMR1
genes. This probemix can also be used to detect deletions/duplications in the aforementioned genes.
The FMR1 (FRAXA)
gene (17 exons) spans ~39 kb on Xq27.3. The AFF2 (FMR2 , FRAXE)
gene (21 exons) spans ~500 kb of genomic DNA and is located in Xq28, at a distance of ~600 kb telomeric of FMR1
. The most common defect in these two genes is an expansion of a trinucleotide repeat sequence in exon 1.
For the FMR1
gene, the trinucleotide repeat motif is CGG, where alleles are classified as normal (unmethylated alleles; 5-44 repeats), intermediate (45-54 repeats), premutation (55-200 repeats) and full-mutation (>200 repeats) (Jorge et al. 2013). Hyper-expansion of FMR1
CGG repeats to >200 is accompanied by hypermethylation of the promoter and gene silencing, which results in Fragile X syndrome (FXS) due to a reduced or absent FMR1
protein (FMRP) (Liu et al. 2021). FXS has an X-linked dominant inheritance pattern and is the most common inherited form of intellectual disability and autism spectrum disorders (Pugin et al. 2017) with a population prevalence of about 1/4000-9000 males and 1/7000-15,000 females (Rzonka et al. 2016). Individuals with a repeat range of 55 to 200 repeats are said to be carriers of a premutation, which does not affect the promoter methylation. However, it can lead to fragile X-associated tremor/ataxia syndrome (FXTAS) affecting primarily men which is characterised by late-onset (>50 years), progressive cerebellar ataxia and intention tremor followed by cognitive impairment. Premutations can also lead to fragile X-associated primary ovarian insufficiency (FXPOI) in females, which manifests as cessation of menstrual periods before reaching the age of 40 (Pugin et al. 2017).
In the AFF2
gene a CCG expansion can occur. In the normal population, the number of repeats varies between 6 and 35 and is increased to >200 in the Fragile XE Syndrome (FRAXE). To what extent the alleles with CCG repeats in the range between 36 and 199 may exhibit a pathogenic role remains elusive. FRAXE is the most prevalent form of non-syndromic X-linked intellectual disability with a milder to borderline phenotype as compared to FXS. It has also been described that microdeletions in AFF2
may be a significant cause of premature ovarian failure (Murray et al. 1999). However, in contrast to FXS, reports of AFF2
full expansions are very rare and the dynamics of this repeat is not as clearly characterized as that of FMR1
leading to poorly defined phenotypes (Jorge et al. 2013).
Results obtained with this SALSA MS-MLPA Probemix ME029 FMR1-AFF2 have shown that FMR1
promoter methylation can be easily detected in DNA samples from males with a FMR1
full mutation (>200 repeats). However, a distinction between premutation alleles and normal alleles cannot be made. For female DNA samples, the presence of an imprinted X-chromosome complicates analysis and can easily result in false positive results. In our studies, the variation in results was too high to reliably detect all full mutation female samples. For methylation quantification
, this product can be used for the analysis of the methylation status of the promoters of FMR1
in male samples
only. Thus, even if it is not possible to measure the length of the repeat by MLPA, the methylation at certain CpG sequences in the promoter region of these genes can still be quantified by MS-MLPA.
More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1384/
The SALSA MS-MLPA Probemix ME029-C1 FMR1-AFF2 contains 48 (MS-)MLPA probes with amplification products between 130 and 472 nucleotides (nt). 20 probes are specific for the FMR1
gene and 14 for the AFF2
gene. Nine of these probes contain an HhaI recognition site and provide information on the methylation status of the exon 1 region of FMR1
(six probes) and AFF2
(three probes). All probes present will also give information on copy number changes in the analysed sample. In addition, ten reference probes are included that are not affected by HhaI digestion and detect genes located outside the Xq27.3 and Xq28 regions. Also, 2 digestion control probes are included in this probemix indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete. Furthermore, two probes with autosomal targets are included for the TGFB1I1
gene (16p11) and one for the PCCB
gene (3p22). Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com
This probemix contains ten quality control fragments generating amplification products between 64 and 118 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and two chromosome Y-specific fragments. More information on how to interpret observations on these control fragments can be found in the MS-MLPA General Protocol and online at www.mrcholland.com