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Published online before print October 2, 2008
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Technical Advance |
From MRC-Holland, Amsterdam, The Netherlands
Abstract
Fragile X syndrome is the most common cause of inherited mental retardation and the second most common cause of mental impairment after trisomy 21. It occurs because of a failure to express the fragile X mental retardation protein. The most common molecular basis for the disease is the abnormal expansion of the number of CGG repeats in the fragile X mental retardation 1 gene (FMR1). Based on the number of repeats, it is possible to distinguish four types of alleles: normal (5 to 44 repeats), intermediate (45 to 54), premutation (55 to 200), and full mutation (>200). Today, the diagnosis of fragile X syndrome is performed through a combination of PCR to identify fewer than 100 repeats and of Southern blot analysis to identify longer alleles and the methylation status of the FMR1 promoter. We have developed a methylation-specific multiplex ligation-dependent probe amplification assay to analyze male fragile X syndrome cases with long repeat tracts that are not amplifiable by PCR. This inexpensive, rapid and robust technique provides not only a clear distinction between male pre- and full-mutation FMR1 alleles, but also permits the identification of genomic deletions, a less frequent cause of fragile X syndrome.
The fragile X mental retardation 1 (FMR1) gene is directly associated with three distinct conditions: fragile X syndrome (the most common X-linked mental retardation syndrome), fragile X-associated tremor/ataxia syndrome, and premature ovarian failure.1, 2 FMR1 encodes the fragile X mental retardation protein, an RNA-binding protein that shuttles from the nucleus to the cytoplasm, where it associates with polyribosomes and suppresses translation of a selective group of mRNAs to which it binds.3, 4 It has been shown that dendrites are subject to fragile X mental retardation protein translation regulation in response to synaptic activity.5 FMR1 is highly conserved across species and contains a CGG trinucleotide repeat sequence of variable length in the 5'-untranslated region of the gene.6 Normal individuals carry 5 to 44 CGG repeats that are usually stably transmitted to the offspring. However, the CGG repeats can become unstable and expand in some families for reasons as yet unknown. Based on the size of the expansion, it is possible to distinguish four types of alleles: normal (5 to 44 repeats), intermediate (45 to 54 repeats), premutation (55 to 200 repeats), and full-mutation (>200 repeats) alleles. Intermediate and premutation alleles are distinguished through repeat instability and family history.7
Both premutation and full-mutation alleles result in FMR1 deregulation: Excess transcription occurs in the premutation setting and is associated with both fragile X-associated tremor/ataxia syndrome and premature ovarian failure; translational suppression of FMR1 mRNA occurs in the full-mutation setting and is strongly linked to fragile X syndrome.8, 9, 10, 11 Fragile X-associated tremor/ataxia syndrome patients display cognitive decline and generalized brain atrophy presenting beyond the fifth decade of life, and approximately 24% of premutation females experience premature ovarian failure. The prevalence of fragile X syndrome is approximately 1 in 4000 males and in 1 in 8000 females; it presents at around 36 months of age as developmental delay and eventually leads to cognitive impairment and other manifestations of the syndrome, such as hyperactivity and anxiety.12 The disease manifests itself most severely in males: IQ values of affected males range between 20 and 70, whereas one-third of female full-mutation patients have an IQ in the mildly retarded range and a corresponding mild-retardation phenotype.2
Although premutation patients exhibit elevated FMR1 mRNA levels, it has been demonstrated that fragile X mental retardation protein expression levels are diminished11 because of reduced translational efficiency presumably caused by the expanded CGG repeat in the 5'-untranslated region of FMR1 mRNA.13 However, it is in the setting of a full mutation that an almost complete absence of fragile X mental retardation protein occurs, and this absence is generally caused by transcriptional silencing of FMR1 through promoter hypermethylation.14, 15 It is noteworthy that FMR1 promoter hypermethylation is absent in premutation patients.
Experimental determination of expansions greater than 
100 repeats is difficult to achieve reliably with standard PCR methodology because of the very high GC content of the target sequence.16, 17
Today, diagnosis of cases not amplifiable by standard PCR is therefore normally performed using Southern blot analysis, a low-resolution, time-consuming technique that requires a large amount of patient genomic DNA. Attempts made through modifications of the PCR technique, such as the use of dimethyl sulfoxide, 7-deaza-dGTP, and combinations of polymerases,18, 19, 20, 21, 22, 23
have produced highly variable results, whereas studies that have investigated the promoter hypermethylation of full-mutation patients, mainly through the methylation-specific PCR technique, have made use of bisulfite-treated DNA, an inefficient transformation that requires large amounts of DNA and frequently does not work with poorer quality or paraffin-derived material.
We have developed a novel assay using the methylation-specific multiplex ligation-dependent probe amplification technique (MS-MLPA).24
MS-MLPA is based on the standard MLPA technique,25
in which up to 50 oligonucleotide probes are hybridized to a denatured DNA sample. However, each MS-MLPA probe is designed to span a recognition site for the methylation-sensitive restriction endonuclease HhaI in its target-specific sequence. After a simultaneous ligation/digestion reaction of the probe/DNA complex using the Ligase-65 enzyme and HhaI, MLPA probes that are hybridized to unmethylated sites are digested and therefore not amplified (Figure 1)
. Comparison with an identical parallel reaction where the digestion step has been omitted reveals both relative copy number and methylation status of the analyzed sample. Here, we report our findings and demonstrate MS-MLPA to be an inexpensive, fast, and robust approach for the analysis of male fragile X syndrome cases that are not resolved by standard PCR.
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Patient Samples
Anonymized DNA samples from fragile X patients (20 full-mutation males and four premutation males) and from four unaffected male controls were generously donated by Dr. Waltraud Friedl (Universitätsklinikum, Bonn, Germany), by Dr. Hans Gille (Free University Medical Centre, Amsterdam, The Netherlands), and by Dr. Flora Tassone (University of California, Davis, CA). The fragile X status of each sample was independently confirmed through PCR/Southern blot analysis (Table 1)
. A commercially available mixture of the DNA of several male individuals, supplied by Promega (Leiden, The Netherlands), was also used as control DNA.
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, and information on probe sequences can be obtained from MRC-Holland (info{at}mlpa.com).
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100 ng of genomic DNA was denatured in a 5 µl Tris-EDTA buffer solution containing 10% glycerol and 20 mM NaOH for 10 minutes at 98°C. After the addition of SALSA MLPA buffer (1.5 µl) and MS-MLPA probes (1.5 µl) to the denatured sample, the mixture was heated for 1 minute at 95°C and for 16 hours at 60°C, allowing the probes to hybridize. After hybridization, a solution containing 3 µl of Ligase buffer A and 10 µl of H2O was added to the mixture at room temperature. The mixture was equally divided into two tubes, and while at 49°C, a mixture of 0.25 µl of Ligase-65, 5 U of HhaI (Promega), and 1.5 µl of Ligase buffer B diluted in H2O to 10 µl was added to one tube, whereas an equivalent mixture without HhaI was added to the second tube. Simultaneous ligation and digestion was then performed by incubation for 30 minutes at 49°C, followed by a 5 minute heat inactivation of the enzymes at 98°C. The ligation products were PCR amplified by the addition of 5 µl of each ligation mixture to a 20 µl PCR mixture containing PCR buffer, dNTPs, SALSA polymerase, and PCR primers (one unlabeled and one Cy5 labeled) at 60°C, followed by 35 cycles of amplification.
Fragment and Data Analysis
PCR products were analyzed using a Beckman-Coulter CEQ 8800 Genetic Analysis System (Beckman-Coulter, Fullerton, CA), and the resulting data analysis was performed using the free Excel-based Coffalyser program, available at http://www.mlpa.com/coffalyser/. Each MS-MLPA reaction was performed at least in duplicate, and samples with incomplete digestion as identified by the digestion control probes were repeated.
Results
Visual inspection of the electropherograms depicted in Figure 2
clearly illustrates the difference in the methylation status of the FMR1 promoter between the three sample categories analyzed in this study. For both the Promega male control DNA and case 10564 (premutation patient), the peaks corresponding to all HhaI-containing probes are absent in the digested electropherograms (Figure 2 B and D)
while remaining present in the undigested samples (Figure 2 A and C)
. This encompasses not only the FMR2 methylation-specific probes (Figure 2
, curved arrows) and digestion control probes (Figure 2
, arrowheads) but also the FMR1 methylation-specific probes (Figure 2
, straight arrows), indicative of lack of methylation in all of these regions. The images for case 4672 (full-mutation patient), however, reveal the presence of FMR1 peaks in both undigested and digested samples (Figure 2 E and F)
, indicative of methylation in the FMR1 promoter region; conversely, the absence of FMR2 and digestion control peaks in the digested sample (Figure 2F)
when compared with its undigested counterpart (Figure 2E)
indicates that these regions remain unmethylated, as expected. Of note, FMR2 methylation-specific probes have been used in this study as internal controls for X chromosome methylation.
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As expected, the analysis showed all of the male unaffected individuals (N = 4, in addition to the Promega male control DNA) and the premutation males (N = 4) to have an FMR1 D:U ratio of <0.05, whereas the full-mutation males (N = 20) presented values ranging from 0.64 to 0.93 (average of 0.82), clearly demonstrating FMR1 promoter hypermethylation (Tables 1
and 3)
. Furthermore, three of the full-mutation males, determined by Southern blot analysis to be mosaics (Table 1)
, were correctly diagnosed by MS-MLPA as having full-mutation alleles. In an additional demonstration of the reliability of the technique in examining male mosaic samples, a dilution series of full-mutation patient 4672 demonstrated that MS-MLPA detected FMR1 promoter hypermethylation in all full-mutation DNA concentrations tested (100, 80, 60, 40, 20, 10, and 5%), attesting the sensitivity of the technique (Table 4)
.
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Conventional PCR-based techniques allow for the accurate detection of only up to 100 CGG repeats in the FMR1 gene. They provide a reliable method for distinguishing normal FMR1 individuals from those carrying intermediate and, in some cases, premutation alleles. When PCR techniques fail, Southern blot analysis is typically the method of choice for differentiating premutation from full-mutation individuals. However, Southern blot analysis requires large amounts of patient DNA and is time consuming. There is therefore a need for the development of a fast and reliable method that enables the distinction between premutation and full-mutation cases, acting as a complement to the standard PCR FMR1 mutation detection and thereby permitting the characterization of all samples within the fragile X syndrome context.
MS-MLPA has previously demonstrated its robustness and reproducibility in a number of previous studies.24, 26, 27, 28
Here, we have used the technique to confirm the mutation status of several fragile X male patients and unaffected controls through analysis of FMR1 promoter methylation. This method relies on the crucial distinction between FMR1 premutation and full-mutation DNA: premutation alleles display no promoter hypermethylation, whereas in full-mutation patients, the promoter region is hypermethylated to varying degrees (Figure 2)
.12, 29
In addition, unlike any other method currently used for fragile X diagnosis, MS-MLPA can reveal the presence of chromosomal aberrations, such as deletions or amplifications, within the investigated loci, because the probe mix used in this study contains several probes spanning the length of FMR1.
Fragile X is significantly more prevalent among males, and typically, the disease is more severe in this group because of the disruption of the only existing copy of FMR1. Our analysis revealed a very clear distinction between male premutation and full-mutation patients, indicative of the robustness of MS-MLPA for diagnosis of this fragile X group. The technique was also attempted on female patients but because of the complexity conferred by the presence an imprinted X chromosome, a small percentage of borderline cases could not be successfully analyzed.
As expected, all of the male unaffected individuals and the male premutation cases showed an FMR1 D:U ratio of <0.05, consistent with the fact that no FMR1 promoter hypermethylation was observed in the sole male X chromosome. By contrast, the 20 full-mutation male patients had an FMR1 D:U ratio average of 0.82, lower than the expected value of 1 but consistent with the mosaicism commonly observed in the FMR1 locus29
and clearly indicative of FMR1 promoter hypermethylation (Tables 1
and 3
; Figure 2
). The three cases determined by Southern blot analysis as displaying mosaicism all presented lower than average FMR1 D:U ratios (Table 1)
.
A further test of the reliability of the technique in detecting a wide range of levels of full-mutation DNA, emulating various mosaicism settings, was successfully performed. In a dilution series of DNA from patient 4672 (full-mutation male), the presence of as little as 5% of full-mutation DNA sufficed to detect FMR1 promoter hypermethylation and thus the presence of full-mutation alleles (Table 4)
.
Noteworthy, one of the premutation cases (241-07) possessed a very high level of CGG repeats (close to 200), indicating the clear distinction in the methylation levels of the FMR1 promoter between premutation and full-mutation alleles, as detected by MS-MLPA.
In the majority of diagnostic laboratories, Southern blot analysis is still the technique of choice to elucidate the number of FMR1 CGG repeats in samples not resolved by conventional PCR. Because of the large amounts of DNA required and because the technique is labor intensive and time consuming, attempts have been made at replacing Southern blot analysis with modifications of the conventional PCR. Results have, however, been highly variable, largely because of the difficulty in amplifying such GC-rich regions. Accurate detection of the fragile X syndrome is crucial, not only because of its relatively high incidence (it is the most common inherited cause of mental retardation), but because of the phenotypical variation of disease presentation, making clinical diagnosis sometimes difficult. In addition, diagnosis of a serious condition such as fragile X syndrome based on a negative PCR result is highly undesirable. Here, we present an inexpensive, fast, and robust technique that provides a clear distinction between premutation and full-mutation FMR1 alleles in male patients, and we therefore conclude that MS-MLPA is a very useful complement to conventional PCR in correctly diagnosing fragile X syndrome in this patient group. Additionally, the methylation level of FMR1 may provide an indication as to the presence of FMR1 mRNA in full-mutation cases, making these patients the target of therapies aimed at increasing translational efficiency, rather than transcriptional activity, of FMR1.
Acknowledgments
We thank Dr. Waltraud Friedl (Universitätsklinikum, Bonn, Germany), Dr. Hans Gille (Free University Medical Centre, Amsterdam, The Netherlands) and Dr. Flora Tassone (University of California, Davis, CA) for the kind donation of patient material.
Footnotes
Address reprint requests to Ralph Carvalho, Willem Schoutenstraat 6, 1057 DN Amsterdam, The Netherlands. E-mail: r.carvalho{at}mlpa.com
Accepted for publication August 18, 2008.
References
imovi S, Vlasic J, Barisic L, Markovic D, Culic V, Pavelic K: A simple and rapid analysis of triplet repeat diseases by expand long PCR. Clin Chem Lab Med 2001, 39:1259-1262[CrossRef][Medline]This article has been cited by other articles:
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  T. Todorov, A. Todorova, A. Kirov, B. Dimitrov, R. Carvalho, A. O H Nygren, I. Boneva, and V. Mitev Fragile X mosaic male full mutation/normal allele detected by PCR/MS-MLPA BMJ Case Reports, May 18, 2009; 2009(may18_1): bcr0620080139 - bcr0620080139. [Abstract] [Full Text]
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