The Principles of Clinical Cytogenetics - Extra Materials - Springer

Genomic Imprinting and Uniparental Disomy 517
methylation could exert its effect on gene transcription by altering interactions between DNA and
nuclear proteins.
The involvement of methylation in the initiation and/or maintenance of genomic imprinting has been
examined extensively. Experiments with transgenic mice, in which a foreign gene is inserted into the
mouse genome by microinjection, have demonstrated that some transgenes show different states of
methylation specific to the parent of origin and that the methylation pattern changes from generation to
generation depending on the sex of the parent transmitting the transgene (33–35). In most cases, a
paternally inherited transgene is less methylated than one that is maternally inherited. In a study of
transgene-bearing elements of the Rous sarcoma virus (RSV) and a fused c-myc gene, the paternally
inherited transgene is undermethylated in all tissues and is expressed only in the heart (35). This observation
suggests that methylation status alone does not determine the expression of a transgene and that
undermethylation might be necessary, but not sufficient, for gene expression. In this same study, the
somatic organs of a male animal with a maternally inherited transgene exhibited a methylated transgene
pattern, but in the testes, the transgene was undermethylated, suggesting that the maternally derived
methylation pattern is eliminated in the testes of male offspring during gametogenesis.
The role of DNA methylation in genomic imprinting is further demonstrated by observations made
in three imprinted endogenous genes in mice: insulin-like growth factor 2 (Igf2), H19 (these two
genes are closely linked on mouse chromosome 7), and the Igf2 receptor gene (Igf2r, on mouse
chromosome 17).
Studies of mouse H19 showed that it is subject to transcriptional regulation by genomic imprinting,
with the maternal allele expressed and the paternal allele silent (36). By comparing CpG methylation
and nuclease sensitivity of chromatin in mouse embryos, Ferguson-Smith et al. (37) showed
that hypermethylation and chromatin compaction in the region of the H19 promoter are associated
with repression of the paternally inherited copy of the gene. This normally silent paternal H19 allele
is activated in DNA methyltransferase-deficient embryos (38), providing in vivo evidence that a
direct correlation is present between DNA methylation and gene activity.
Studies of the mouse Igf2 gene showed that, contrary to H19, the paternal allele is expressed in
embryos, whereas the maternal allele is silent, but both parental alleles are transcriptionally active in
the choroid plexus and leptomeninges (39). Therefore, imprinting of Igf2 might also be tissue-specific.
In addition, studies using mouse embryos with maternal duplication and paternal deficiency of the
region of chromosome 7 that encompasses Igf2 showed that the chromatin of the 5' region of the
repressed maternal Igf2 allele is potentially active for transcription, that is, it is hypomethylated and
contains DNase I hypersensitive sites (40). Recently, a region of paternal-specific methylation
between H19 and Igf2 has been postulated to function as the imprint control region. This imprint
control region, when unmethylated, acts as a chromatin boundary or insulator that blocks the interaction
of Igf2 with its enhancer, thus resulting in silencing of the Igf2 gene, as is observed on the
maternal chromosome. On the paternal chromosome, this region is methylated, resulting in the loss
of enhancer-blocking activity and allowing the expression of Igf2 (41,42). A deletion within this
imprint control region results in loss of imprinting of both H19 and Igf2.
Studies of the mouse Igf2r gene indicated that the maternal allele is expressed and the paternal
allele is silent (43). The parental-origin-specific difference in methylation for this gene has been
demonstrated in two distinct CpG islands (44). Here, while the promoter is methylated on the inactive
paternal allele, an intronic CpG island is methylated only on the expressed maternal allele, suggesting
that methylation of the latter site is necessary for expression of the Igf2r gene.
In humans, the methylation patterns of the parental alleles have been determined for several
imprinted loci on chromosome 15 at bands 15q11-q13. These include the ZNF127/DN34 gene
(D15S9) studied in PWS and AS patients (45) and in complete hydatidiform moles (46), the small
nuclear ribonucleoprotein polypeptide N (SNRPN) gene (47,48), and the DNA sequence PW71
(D15S63) (49). Distinct differences in methylation of the parental alleles are observed in all instances.
This is also true for some of the other known imprinted genes in humans: H19 (maternal allele active)