Classification of Epimutations

It is obvious that errors in replicating the DNA methylation pattern and/or the histone pattern can affect the epigenetic state of a gene in the daughter cell. Such epimutations (Holliday 1987) can lead to inappropriate activation of a gene that should be silent, or inactivation of a gene that should be active. Epimutations can also result from mutations in cis-regulatory elements or trans-acting factors. In the following, we will refer to epimutations that occur without any DNA sequence change as primary or "true" epimutations, and to epimutations that result from a DNA mutation as secondary epimutations.

Secondary Epimutations

Secondary epimutations are most often the result of a hereditary DNA sequence change and present in all cells of a patient. The underlying genetic defect can be close to the affected gene (cis) or impair the function of an epigenetic protein encoded somewhere else in the genome (trans).

Secondary Epimutations Resulting from a c/s-Acting DNA Mutation

There are at least two genetic diseases in which a secondary epimutation represents the major pathogenetic mechanism. These are the fragile X mental retardation syndrome (FMR1) and the facioscapulohumeral muscular dystrophy (FSHD). FMR1 is an X-linked dominant disease caused by the expansion of an unstable trinucleotide repeat (CGG) within exon 1 of the FMR1 gene. It is one of the most common causes of mental retardation. The number of repeats varies in the human population. Repeats with more than 58 copies are unstable and can expand to several hundred copies during the proliferation of the diploid oogonia in the fetal ovary. After fertilization of an oocyte carrying an expanded FMR1 allele, the CGG repeat and FMR1 promoter are methylated. DNA methylation, histone deacetylation and the establishment of repressive chromatin in this region silence the FMR1 gene.

FSHD is an autosomal dominant disorder that has been linked to a 3.3-kb tandemly repeated sequence (D4Z4) in the subtelomeric region of the long arm of chromosome 4. In normal individuals the number of D4Z4 repeats varies between 11 and 150 units, whereas FSHD patients have fewer than 11 repeats. Gabellini et al. have shown that a sequence element within D4Z4 specifically binds a multiprotein complex consisting of the transcriptional repressor YY1, the architectural protein HMGB2 and nucleolin, and that this multiprotein complex mediates transcriptional repression of adjacent genes in 4q35 genes (Gabellini et al. 2002), probably by establishing a repressive chromatin structure over a very large distance. Based upon these results, the authors propose that deletion of D4Z4 is associated with an open chromatin structure in 4q35 and the inappropriate expression of several genes within this region. However, these findings have remained controversial.

Imprinting defects resulting from mutations in a cz's-acting imprinting control element are another example of secondary epimutations. Imprinting is an epigenetic process by which the male and the female germline mark specific chromosome regions so that only the maternal or paternal allele of certain genes is active. Imprint establishment and imprint maintenance are under the control of imprinting centres (IC). The IC on human chromosome 15 contains two critical elements, which are defined by the shortest region of deletion overlap (SRO) in Angelman syndrome (AS) and Prader-Willi syndrome (PWS) patients with an imprinting defect (AS-SRO and PWS-SRO, respectively) (Buiting et al. 1995). The AS-SRO element is necessary for the establishment of the maternal imprint in the female germline. A deletion of this element prevents maternal imprinting of the mutated chromosome. A child inheriting this chromosome will develop AS, which is a neurogenetic syndrome characterized by severe mental retardation, lack of speech, jerky movements and a happy disposition (estimated prevalence 1/15,000 newborns). It is caused by the loss of function of the UBE3A gene, which encodes an enzyme involved in targeted protein degradation. In the brain, the gene is active on the maternal chromosome only. In contrast to many other imprinted genes, mono-allelic expression of UBE3A is not associated with differential DNA methylation of the promoter/exon 1 region. There is some tentative evidence that the paternal allele is silenced by an antisense RNA which originates at the neighbouring SNRPN locus (Rougeulle et al. 1998; Runte et al. 2001). In normal individuals, SNRPN is methylated on the maternal chromosome and expressed from the paternal chromosome (Ozce-lik et al. 1992; Zeschnigk et al. 1997). In AS patients with an imprinting defect (which accounts for approximately 3% of cases), the maternal SNRPN allele is unmethylated and expressed, and the maternal UBE3A allele is silenced. Of these patients, 10% have an AS-SRO deletion, whereas 90% have a primary epimutation (see Sect. 2.2). Most of the other AS patients have a large maternally derived chromosomal deletion, a maternal UBE3A mutation or paternal uniparental disomy 15.

The PWS-SRO of the chromosome 15 IC is necessary for the postzygotic maintenance of the paternal imprint (Bielinska et al. 2000). A paternally derived deletion of this element leads to an epigenetic state that resembles the maternal imprint. A child with such a chromosome will develop PWS, which is characterized by neonatal muscular hypotonia, hypogonadism, hyperphagia and obesity, short stature, small hands and feet, sleep apnoea, behavioural problems and mild to moderate mental retardation (estimated prevalence, 1/25,000 newborns). PWS is caused by the loss of function of imprinted genes which are active on the paternal chromosome only. Although all of the genes in the critical region are known, it is unclear which are the "PWS genes". In patients with an imprinting defect, which is found in approximately 1% of cases, all paternally expressed genes are silent. Of these patients, 10% have a PWS-SRO deletion, whereas 90% have a primary epimutation (see Sect. 2.2). Almost all of the other PWS patients have a large paternally derived chromosomal deletion, or maternal uniparental disomy.

Most of the IC deletions are familial deletions. Since deletions of the AS-SRO affect maternal imprinting only, they are silently transmitted through the paternal germline. Likewise, deletions of the PWS-SRO, which affect the paternal imprint only, are silently transmitted through the maternal germline. This explains why in some families only a few and distantly related individuals are affected. In some cases, the IC deletion has occurred de novo or is the result of germline mosaicism. There is only one case in which the deletion occurred postzygotically (Bielinska et al. 2000).

In contrast to AS and PWS, more than 50% of patients with transient neonatal diabetes mellitus or Beckwith-Wiedemann syndrome (BWS) have an imprinting defect. BWS is an overgrowth syndrome characterized by high birth weight, hypoglycaemia, macroglossia, exomphalos and increased risk of Wilms' tumour (estimated prevalence, 1/25,000 newborns). It is caused by overexpression of the paternally active IGF2 gene and silencing of the maternally expressed H19 gene or by silencing of the maternally active CDKN1C gene. These genes map to the short arm of chromosome 11, but are controlled by two different ICs, the IGF2/H19 IC (IC1) and LIT1/KCNQ1OT1 (IC2), which controls imprinting of CDKN1C. Similar to imprinting defects in AS and PWS, secondary epimutations in BWS are very rare. Sparago et al. have recently identified two families segregating a microdeletion in the IGF2/H19 IC. Maternal transmission of the deletions resulted in hypermethylation of the IGF2/H19 IC, biallelic IGF2 expression, H19 silencing and BWS (Sparago et al. 2004). Prawitt et al. (2005) have identified a family with a similar deletion. A deletion of IC2 has been described by Niemitz and colleagues (2004). When inherited maternally, the deletion caused BWS with silencing of CDKN1C. When inherited paternally, there is no phenotype, suggesting that the LIT1/KCNQ1OT1 RNA itself is not necessary for normal development in humans.

A unique epimutation affecting the a-globin gene HBA2 has recently been described by Tufarelli and colleagues (2003). The authors studies an individual with an inherited form of a-thalassaemia who has a deletion that results in a truncated, widely expressed gene (LUC7L) becoming juxtaposed to the structurally normal a-globin gene HBA2. Although it retains all of its local and remote cz's-regulatory elements, expression of HBA2 is silenced. LUC7L is transcribed from the opposite strand to the a-globin genes. In the patient, RNA transcripts from the truncated copy of LUC7L (missing the last three exons) extend into the HBA2 CpG island, thus generating antisense transcripts with respect to HBA2. Antisense RNA transcription appears to mediate methylation of the HBA2 CpG island during early development and silencing of HBA2 expression.

There are also several examples of chromosomal translocations affecting the epigenetic state of genes adjacent to the breakpoints. This is in particular thecaseintranslocationsinvolvingtheXchromosome. Averyinstructivecase was published by Jones and colleagues (1997). The authors studied a male patient with an unbalanced X;13 translocation [46,XY,der(13)t(X;13)(q10q10)] and bilateral retinoblastoma. DNA replication and methylation studies suggested that the extra copy of Xq, which is attached to the long arm of one chromosome 13, was inactivated and that inactivation had spread to chromosome 13 and silenced the RBI gene in 13q14. This epimutation is equivalent to a constitutional RBI mutation and explains the development of bilateral tumours in this patient.

Secondary Epimutations Resulting from trans-Acting DNA Mutations

In the last few years, many epigenetic players have been identified. They include DNA methyltransferases, methyl-CpG binding proteins, histone modifying enzymes, chromatin-remodelling factors and others. Loss of function of these proteins has a major impact on the epigenetic control of gene expression. In contrast to epimutations caused by a cz's-acting DNA mutation, epimutations causedby trans-actingDNA mutations can affect many different genes on different chromosomes. In humans, several recognizable syndromes have been linked to a mutation in one of the epigenetic players. Mutations in the de novo DNA methyltransferase DNMT3a, for example, cause autosomal-recessive ICF syndrome (immunodeficiency, centromere instability and facial anomalies). The patients die of severe recurrent infections. Chromosome instability correlates with severe hypomethylation of the satellite DNA.

X-linked a-thalassaemia mental retardation (ATRX) syndrome is a developmental disorder characterized by mental retardation, facial dysmorphism, abnormal genitalia and anaemia resulting from reduced expression of the a-globin genes. It results from mutations in the ATRX gene, which encodes a member of the SWI/SNF family of chromatin remodelling factors.

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder. Girls with RTT have apparently normal development throughout the first 6 months of life; they subsequently begin to loose previously acquired skills and develop microcephaly, hand stereotypies, autistic features, seizures and gait apraxia. RTT is caused by mutations in the MeCP2 gene, which encodes the methyl-CpG-binding protein 2. One function of MeCP2 is to recruit the Sin3A corepressor complex, which contains histone deacetylase, and to set up repressive chromatin. Initially it was believed that the loss of MeCP2 leads to widespread loss of gene repression. This, however, does not appear to be the case. To date, only two genes (BDNF and DLX5) have been identified as target genes (Chen et al. 2003; Horike et al. 2005).

Primary or "True" Epimutations

Compared to secondary epimutations, primary epimutations appear to be more frequent. As mentioned above, only 10% of imprinting defects in AS and PWS are caused by an IC mutation; in 90% of cases the imprinting defect is a primary epimutation. In BWS almost all imprinting defects are primary epimutations. Epimutations affecting genomic imprints can occur during imprint erasure in primordial germ cells, imprint establishment during later stages of gametogenesis or imprint maintenance after fertilization. If it occurs in the germline, all cells of the patient are affected. If it occurs after fertilization, it often results in somatic mosaicism.

Buiting et al. have found that in PWS patients with an imprinting defect not caused byanIC mutation the affected chromosome is always derived from the paternal grandmother (Buiting et al. 2003). This finding suggests that the (grand)maternal imprint was not erased in the paternal germline. Thus, the child inherited an epigenetic state from the grandmother. This is the best example of transgenerational epigenetic inheritance in man.

Wey et al. have recently described a PWS patient with a mosaic imprinting defect (Wey et al. 2004). In this patient, the epimutation most likely occurred after fertilization, although we cannot exclude the possibility that somatic mosaicism results from the postzygotic correction of an inherited epimutation.

In contrast to PWS, mosaic imprinting defects in AS are relatively common. Nazlican et al. have estimated that at least 30% of AS patients with a primary imprinting defect are mosaics (Nazlican et al. 2004). In two patients studied, somatic mosaicism was proved by molecular and cellular cloning, respectively. X inactivation studies of cloned fibroblasts from one patient suggest that the imprinting defect occurred before the blastocyst stage. To quantify the degree of mosaicism, the authors developed a quantitative methylation assay based on real-time PCR. In 24 patients tested, the percentage of normal cells ranged from less than 1% to 40%. Regression analysis suggested that patients with a higher percentage of normally methylated cells tend to have milder clinical symptoms than patients with a lower percentage. Some mosaic patients have "atypical Angelman syndrome" characterized by obesity, muscular hypotonia and ability to speak (Gillessen-Kaesbach et al. 1999). We might assume that the role of mosaic imprinting defects on chromosome 15 in mental retardation is underestimated.

Primary epimutations have not only been recognized in "imprinting disorders", but also in cancer. In 1983, A. Feinberg and B. Vogelstein discovered altered DNA methylation in cancer cells (Feinberg and Vogelstein 1983). Subsequently, these and other authors demonstrated that hypomethylation can lead to inappropriate activation of oncogenes. In 1986, S. Baylin and colleagues identified hypermethylation of the calcitonin gene in human lung cancers and lymphomas (Baylin et al. 1986), but the role of these changes in tumour development were unknown. Soon after the discovery of the first tumour suppressor gene (the retinoblastoma gene RBI), our own group found that the RBI promoter is methylated in a significant subset of retinoblastomas (Greger et al. 1989, 1994), suggesting that tumour-suppressor silencing can also occur by an epigenetic pathway. Subsequently, methylation of tumour-suppressor genes has been found in virtually all tumours, and the field of cancer epigenetics is rapidly growing (Feinberg and Tycko 2004).

In general, tumour-associated epimutations are found only in premalig-nant or malignant cells. There is only one case in which an inherited cancer epimutation has been described. Suter et al. have reported two individuals with soma-wide, allele-specific and mosaic hypermethylation of the DNA mismatch repair gene MLH1 (Suter et al. 2004). Both individuals lacked evidence of DNA sequence mutation in any mismatch repair gene, but had multiple primary tumours that show mismatch repair deficiency. The epimutation was also present in spermatozoa of one of the individuals, indicating a germline defect and the potential for transmission to offspring.

Primary epimutations appear to play a role in cardiovascular disease also. Similar to tumours, atherosclerotic lesions are characterized by global DNA hypomethylation and local DNA hypermethylation. These similarities should not be surprising, because a key step of the atherogenetic process is the proliferation and migration of smooth muscle cells. Once within the intima, the phenotype of the smooth muscle cells switches from contractile to "dedif-ferentiated". It has been suggested that methylation of oestrogen receptor-a gene (ESR1) could contribute to these processes (Ying et al. 2000).

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