Introduction
The possibility of detecting mutations in MECP2 in patients with Rett syndrome has changed the face of this unique disorder and has accelerated research in this field. Several articles have already been written about the genetics of Rett syndrome. In this article, we do not attempt to discuss the genetics of Rett syndrome in great detail but instead summarize knowledge that is important for clinicians caring for these patients and their parents.
Description of the First Mutations in MECP2
It had been speculated for many years that genetic defects in the X chromosome are involved in the pathogenesis of Rett syndrome because almost only female individuals are affected. It was assumed that there is male lethality in this condition. However, because greater than 99% of Rett syndrome cases are sporadic, linkage studies were not possible until 1998, when a family identified with a maternal inheritance pattern of Rett syndrome permitted exclusion mapping studies to be performed that then defined chromosome Xq28 as a candidate region for the Rett syndrome gene.[1] In 1999, Amir and colleagues identified the first mutations in MECP2 in 5 of 21 sporadic cases with Rett syndrome.[2] MECP2 was not one of the first candidate genes on chromosome Xq28 that were investigated because it is expressed in all tissues and had no known brain-specific function.[3]
MECP2 and the MECP2 Protein
The MECP2 gene that is mutated in Rett syndrome is located on the long arm of the X chromosome at Xq28 and is subject to X-chromosome inactivation.[4,5] The gene consists of four exons that code for two different isoforms of the MeCP2 protein. Until early 2004, it was thought that only one isoform existed. For this isoform, a start codon in exon 2 is used and exon 1 is noncoding, explaining why, until recently, exon 1 was not analyzed in the routine mutation screening. However, it has since been found that there is a second isoform of the protein in which exon 1 includes coding sequences and exon 2 is eliminated by alternative splicing (Figure 1). Unexpectedly, this new protein isoform is far more abundant than the one originally described.[6,7] The protein isoform that was described first was subsequently named MECP2e2, and the newly described isoform was named MeCP2e1.
The MeCP2 protein was first described in 1992.[8] It is one of five known proteins sharing a methyl-CpG binding domain that allows them to bind to methylated CpGs in the DNA. Through different mechanisms, this binding leads to a transcriptional repression of genes in the area of these binding sites. The exact mechanisms of this suppression are the topic of other articles in this issue. To understand the significance of the position and type of mutation, it is important to know that there are two, possibly three, domains of the protein that are essential for its function. The methyl-CpG binding domain is encoded by exons 3 and 4 and binds to the methylated DNA (Figure 2).[9] The second important domain is the transcriptional repression domain, which contains binding sites for the corepressor complex and is therefore involved in the repression process.[10] The third domain is located in the C-terminus of the protein and is meant to facilitate the binding both to naked DNA and to the nucleosome core.[11] However, this domain is not yet well defined.
MECP2 and the MECP2 Protein
The MECP2 gene that is mutated in Rett syndrome is located on the long arm of the X chromosome at Xq28 and is subject to X-chromosome inactivation.[4,5] The gene consists of four exons that code for two different isoforms of the MeCP2 protein. Until early 2004, it was thought that only one isoform existed. For this isoform, a start codon in exon 2 is used and exon 1 is noncoding, explaining why, until recently, exon 1 was not analyzed in the routine mutation screening. However, it has since been found that there is a second isoform of the protein in which exon 1 includes coding sequences and exon 2 is eliminated by alternative splicing (Figure 1). Unexpectedly, this new protein isoform is far more abundant than the one originally described.[6,7] The protein isoform that was described first was subsequently named MECP2e2, and the newly described isoform was named MeCP2e1.
The MeCP2 protein was first described in 1992.[8] It is one of five known proteins sharing a methyl-CpG binding domain that allows them to bind to methylated CpGs in the DNA. Through different mechanisms, this binding leads to a transcriptional repression of genes in the area of these binding sites. The exact mechanisms of this suppression are the topic of other articles in this issue. To understand the significance of the position and type of mutation, it is important to know that there are two, possibly three, domains of the protein that are essential for its function. The methyl-CpG binding domain is encoded by exons 3 and 4 and binds to the methylated DNA (Figure 2).[9] The second important domain is the transcriptional repression domain, which contains binding sites for the corepressor complex and is therefore involved in the repression process.[10] The third domain is located in the C-terminus of the protein and is meant to facilitate the binding both to naked DNA and to the nucleosome core.[11] However, this domain is not yet well defined.
Mutation Detection in MECP2
In Rett syndrome, greater than 99% of patient mutations are sporadic. Not surprisingly, more than 200 different MECP2 mutations have been described so far. As in other genetic disorders with such a variety of possible mutations, direct sequencing of the coding and splice-site regions is the method most laboratories use. It is important to note that exon 1 was not initially included in the routine screening program for mutations in MECP2 because it was thought to be noncoding. This has changed since the description of MeCP2e1, the new isoform of MeCP2, but it remains controversial as to how frequent mutations in exon 1 are. Mnatzakanian and coworkers described deletions involving exon 1 in 2 of 19 patients with Rett syndrome for whom sequencing of the other regions had not revealed any mutations, whereas Evans and coworkers did not detect any mutations in 97 patients.[6,12] Despite this, the analysis of exon 1 is considered part of the routine sequencing program for MECP2, performed in all patients with clinically defined Rett syndrome and especially in those for whom previously performed analyses have not revealed any mutations in the other exons.
In a more generalized screening approach for the identification of MECP2 mutations in large groups of patients with nondefined mental retardation or other disorders, such as autism, denaturing high-performance liquid chromatography and single-strand conformational polymorphism analyses have been applied successfully.[13-15] Thistlethwaite and coworkers applied an electronic DNA microchip using serial differential hybridization to detect the eight most frequent MECP2 mutations.[16]
Several methods have been applied to detect gross rearrangements of MECP2, including multiplex ligation-dependent probe amplification, gene dosage analysis by real-time quantitative polymerase chain reaction, and fluorescent in situ hybridization.[17-20] Although it is still unclear which is the most reliable, a routine mutation screen of MECP2 should include one of these methods.
Mutations in MECP2
Thus far, more than 200 mutations in MECP2 have been described.[21] To facilitate genotype-phenotype correlation analyses, the locus-specific International Rett Syndrome Association MECP2 variation database has been established (http://mecp2.chw.edu.au/). Eight MECP2 mutation hot spots are located at CpG dinucleotides, all transitions from cytosine to thymine. They comprise 65% of all mutations.[21] The other MECP2 mutations are less frequent; some have been found only once or twice. Although the mutations can be found in all parts of the gene, there is an obvious pattern in their distribution. Most missense mutations are located in the methyl-CpG binding domain and in the last part of the transcriptional repression domain, whereas the nonsense mutations are located mainly between the methyl-CpG binding and transcriptional repression domains and in the first part of the transcriptional repression domain.
In some cases of Rett syndrome, it is difficult to decide whether the nucleotide change found is of pathogenic significance or if it is only one of the several polymorphisms present in MECP2. For some patients, it is helpful to consult the International Rett Syndrome Association MECP2 variation database to learn whether other patients with Rett syndrome with the same nucleotide change have already been described. For other patients, it is informative to investigate the extended family. However, sometimes it remains unclear whether the nucleotide change explains the clinical picture.
It was noted early that there is a region at the C-terminus that harbors a hot spot for deletions (see Figure 2). The proximal breakpoint of these deletions, which can be found in about 10% of patients, is located in a section with repetitive sequence elements between nucleotides 1050 and 1200.[22] Furthermore, many of the larger rearrangements have one breakpoint in this region.[18] They were detected in 16% of patients with classic Rett Syndrome in whom the sequencing had not revealed any mutations.[18]
In the literature, the reported detection rate for MECP2 mutations in patients with clinically defined Rett syndrome is between 60% and 80%. It is obvious that these rates are influenced by the clinical parameters used to select patients for genetic analysis. In our experience, the detection rate in girls with classic Rett syndrome is much higher than reported: detection approaches 95% if the analysis includes exon 1 and the search for large deletions.
Functional Consequences of Mutations in MECP2
The methyl-CpG binding domain of MeCP2 is responsible for the binding to the methylated DNA. Consequently, mutations affecting the methyl-CpG binding domain can be expected to interfere with this binding. Yusufzai and coworkers have shown that missense mutations in this area indeed impair the selectivity for methylated DNA, whereas one of the few nonsense mutations affecting the methyl-CpG binding domain leads to an inability of the protein to bind to DNA altogether.[23] Truncating mutations affecting the transcriptional repression domain lead to a protein that binds to methylated DNA but fails to repress the transcription.[23] They also found that a truncated protein is being degraded faster than wild-type proteins.
Spectrum of Phenotypes With Mutations in MECP2 in Girls
The detection of mutations in MECP2 enables us to confirm the diagnosis of Rett syndrome in cases that do not fulfill all diagnostic criteria, which is important because the spectrum of phenotypes is wide. For example, some girls with Rett syndrome never learn to roll over, sit, walk, or talk, but others are able to sing songs and run, and they have good hand function.[24] Also, some girls with Rett syndrome are born with a microcephaly at birth or a macrocephaly.[24,25]
The speculation that other disorders are caused by mutations in MECP2 has not been proved conclusive. Although some autism cases have been reported, large studies have ruled out MECP2 mutations as playing a major role in the pathogenesis of autism.[14,26] The same seems to be true for patients with Angelman syndrome. Single cases with mutations in MECP2 have been described, but in larger studies, these mutations could not be confirmed in a large proportion of patients with Angelman syndrome.[27,28]
Boys With Mutations in MECP2
A confounding feature of Rett syndrome is that it almost exclusively affects girls; it was previously thought that Rett syndrome is an X-linked disorder with male lethality. However, there was never evidence of this in families affected with Rett syndrome. Studies in patients with Rett syndrome revealed that mutations are almost exclusively of paternal origin.[29] The mutations in MECP2 are obviously a product of spermatogenesis, because the father provides an X chromosome only to his daughters, not to his sons; therefore, boys are not affected.
To date, about 60 male patients with mutations in MECP2 have been reported, and they can be separated clinically into three groups:
Mutations that are found in female patients with Rett syndrome. They show a severe epileptic encephalopathy with frequent apneas. Most of them have been identified because their sisters had classic Rett syndrome. In these cases, the mutation is passed on maternally, not paternally. This seems to be the phenotype of a male patient with a typical Rett syndrome mutation.[30-34]
Mosaic form mutations or XXY Klinefelter syndrome with mutations found in female patients with Rett syndrome. They clinically resemble female patients with Rett syndrome, indicating that the typical Rett syndrome phenotype can develop when only about 50% of all cells express the defective gene.[31,32,35-38]
Mutations not frequently found in female patients with Rett syndrome. The boys in this group present with symptoms that do not resemble Rett syndrome. Most have unspecific, nonprogressive mental retardation; some present with varying neurologic and psychiatric symptoms.[39-46] Overall, this group of patients is not well defined, and for some mutations, the pathogenetic effect has been questioned.[21]
Conclusion
Until 1999, the diagnosis of Rett syndrome was based solely on the clinical picture because no biochemical or genetic markers were known. Through molecular analyses, we have learned that the clinically defined Rett syndrome diagnosis, based on the established criteria, is very reliable because the vast majority of female patients with classic Rett syndrome have mutations in MECP2. In fact, the mutation detection rate in this group is so high that one could question if mutation analysis of MECP2 should even be performed in a typical case. We have also learned that mutations can be found in girls with a clinically incomplete picture of Rett syndrome. With an increasing number of atypical Rett syndrome cases being reported, clinicians have to ask themselves whether MECP2 analysis should be performed in all patients with undefined mental retardation. However, at least in girls, the data gathered so far do not support that approach. Although there have been single reports of female patients with autism or Angelman syndrome who carry mutations in MECP2, larger studies have not provided evidence that MECP2 mutations play a major role in the pathogenesis of these disorders. Altogether, the spectrum of clinical phenotypes in female patients with mutations in MECP2 is wide; there is no current convincing evidence that these mutations are the primary defect for other neurologic disorders. Therefore, the indication for genetic analysis in female patients can be based on the established diagnostic criteria, keeping in mind that not all of them have to be fulfilled. A clinical checklist should be used to minimize the amount of negative genetic test results.[47]
In boys, MeCP2 mutations seem to be very rare and the clinical phenotype is not well defined. It seems unquestionable that all boys with a Rett syndrome phenotype should be tested for mosaic mutations or an XXY genotype (group 2). It also appears reasonable to test boys with a severe encephalopathy and frequent apneas (group 1). In contrast, we do not have enough data available to search for MECP2 mutations in boys from group 3. The phenotypes described up to now are unspecific and too frequent in the mentally retarded population to allow for sensible mutation screening outside research programs.
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