Epigenetics and developmental programming of welfare and production traits in farm animals.

The concept that postnatal health and development can be influenced by events that occur in utero originated from epidemiological studies in humans supported by numerous mechanistic (including epigenetic) studies in a variety of model species. Referred to as the 'developmental origins of health and disease' or 'DOHaD' hypothesis, the primary focus of large-animal studies until quite recently had been biomedical. Attention has since turned towards traits of commercial importance in farm animals. Herein we review the evidence that prenatal risk factors, including suboptimal parental nutrition, gestational stress, exposure to environmental chemicals and advanced breeding technologies, can determine traits such as postnatal growth, feed efficiency, milk yield, carcass composition, animal welfare and reproductive potential. We consider the role of epigenetic and cytoplasmic mechanisms of inheritance, and discuss implications for livestock production and future research endeavours. We conclude that although the concept is proven for several traits, issues relating to effect size, and hence commercial importance, remain. Studies have also invariably been conducted under controlled experimental conditions, frequently assessing single risk factors, thereby limiting their translational value for livestock production. We propose concerted international research efforts that consider multiple, concurrent stressors to better represent effects of contemporary animal production systems.

Fragile-X syndrome and myotonic dystrophy: parallels and paradoxes.

Fragile-X syndrome and myotonic dystrophy are caused by triplet repeat expansions embedded in CpG islands in the transcribed non-coding regions of the FMR1 and the DMPK genes, respectively. Although initial reports emphasized differences in the mechanisms by which the expanded triplet repeats caused these diseases, results published in the past year highlight remarkable parallels in the likely molecular etiologies. At both loci, expansion is associated with altered chromatin, aberrant methylation, and suppressed expression of the adjacent FMR1 and DMAHP genes, implicating epigenetic mediation of these genetic diseases.

p16INK4a promoter is hypermethylated at a high frequency in esophageal adenocarcinomas.

Loss of heterozygosity (LOH) of 9p21, which contains the p16INK4a tumor suppressor gene locus, is one of the most frequent genetic abnormalities in human neoplasia, including esophageal adenocarcinomas. Only a minority of Barrett's adenocarcinomas with 9p21 LOH have a somatic mutation in the remaining p16 allele, and none have been found to have homozygous deletions. To determine whether p16 promoter hypermethylation may be an alternative mechanism for p16 inactivation in esophageal adenocarcinomas, we examined the methylation status of the p16 promoter in flow-sorted aneuploid cell populations from 21 patients with premalignant Barrett's epithelium or esophageal adenocarcinoma. Using bisulfite modification, primer-extension preamplification, and methylation-specific PCR, we demonstrate that the methylation assay can be performed on 2 ng of DNA (approximately 275 cells). Eight of 21 patients (38%) had p16 promoter hypermethylation and 9p21 LOH, including 3 patients who had only premalignant Barrett's epithelium. Our data suggest that promoter hypermethylation with LOH is a common mechanism for inactivation of p16 in the pathogenesis of esophageal adenocarcinomas.

Deletion and reduced expression of the Fanconi anemia FANCA gene in sporadic acute myeloid leukemia.

Fanconi anemia (FA) is an autosomal recessive chromosomal instability disorder caused by mutations in one of seven known genes (FANCA,C,D2,E,F,G and BRCA2). Mutations in the FANCA gene are the most prevalent, accounting for two-thirds of FA cases. Affected individuals have greatly increased risks of acute myeloid leukemia (AML). This raises the question as to whether inherited or acquired mutations in FA genes might be involved in the development of sporadic AML. Quantitative fluorescent PCR was used to screen archival DNA from sporadic AML cases for FANCA deletions, which account for 40% of FANCA mutations in FA homozygotes. Four heterozygous deletions were found in 101 samples screened, which is 35-fold higher than the expected population frequency for germline FANCA deletions (P<0.0001). Sequencing FANCA in the AML samples with FANCA deletions did not detect mutations in the second allele and there was no evidence of epigenetic silencing by hypermethylation. However, real-time quantitative PCR analysis in these samples showed reduced expression of FANCA compared to nondeleted AML samples and to controls. These findings suggest that gene deletions and reduced expression of FANCA may be involved in the promotion of genetic instability in a subset of cases of sporadic AML.

The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus.

T-associated maternal effect (Tme) is the only known maternal-effect mutation in the mouse. The defect is nuclear-encoded and embryos that inherit a deletion of the Tme locus from their mother die at day 15 of gestation. There are many genomically imprinted regions known in the mouse genome but so far no imprinted genes have been cloned. The Tme locus is absent in two chromosome-17 deletion mutants, Thp and the tLub2, and its position has been localized using these deletions to a 1-cM region. We report here that the genes for insulin-like growth factor type-2 receptor (Igf2r) and mitochondrial superoxide dismutase-2 (Sod-2) are absent from both deletions. Probes for these genes and for plasminogen (Plg) and T-complex peptide 1 (Tcp-1) were used in pulsed-field gel mapping to show that Tme must lie within a region of 800-1,100 kb. We also demonstrate that embryos express Igf2r only from the maternal chromosome, and that Tcp-1, Plg and Sod-2 are expressed from both chromosomes. Therefore Igf2r is imprinted and closely linked or identical to Tme.

Epigenetic variation illustrated by DNA methylation patterns of the fragile-X gene FMR1.

Genomic methylation patterns of mammals can vary among individuals and are subject to dynamic changes during development. In order to gain a better understanding of this variation, we have analyzed patterns of cytosine methylation within a 200 bp region at the CpG island of the human FMR1 gene from leukocyte DNA. FMR1 is normally methylated during inactivation of the X chromosome in females and it is also methylated and inactivated upon expansion of CGG repeats in fragile-X syndrome. Patterns of methylation (epigenotypes) were determined by the sequencing of bisulfite-treated alleles from normal males and females and alleles from a family of five brothers who are methylation mosaics and are affected to various degrees by the fragile-X syndrome. Our data indicate that: (i) methylation of individual CpG cytosines is strikingly variable in hypermethylated epigenotypes obtained from a single individual, suggesting that maintenance of cytosine methylation is a dynamic process; (ii) methylation of non-CpG cytosines in the region studied may occur but is rare; (iii) mosaicism of methylation in the analyzed fragile-X males is remarkably similar to that found for the active X and inactive X alleles in normal females, suggesting that the methylation mosaicism of some fragile-X males reflects similar on and off states of FMR1 expression that exist in normal females; (iv) hypermethylation is slightly more pronounced on fragile-X alleles than on normal inactive X alleles of females; (v) the general dichotomy of hypo- and hypermethylated alleles persisted over the 5 year period that separated samplings of the fragile-X males; (vi) methylation variability was most pronounced at a consensus binding sequence for the alpha-PAL transcription factor, a sequence that may play a role in regulating expression of FMR1.

The mouse Rsk3 gene maps to the Leh66 elements carrying the t-complex responder Tcr.

A variant form of mouse Chromosome (Chr) 17, the t-haplotype, contains several loci responsible for transmission ratio distortion in males. Sperm carrying the responder locus (Tcr) have a high probability of fertilizing eggs at the expense of wild-type sperm, provided that distorter loci (Tcd-1 to Tcd-5) are expressed during spermatogenesis. Tcr has been mapped to the Leh66b region within a maximum of 155 kb. In the search for genes in the genomic region Leh66EI, we have identified the mouse homolog of human ribosome S6 kinase 3 (RSK3) on cosmid DNA. The complete mouse Rsk3 gene is encoded in the region Leh66a of t-haplotypes and Leh66EI of the wild-type chromosome. It consists of at least 13 exons spanning over more than 120 kb. Rsk3 is expressed in embryos and in several adult organs including testis. Cosmids covering 100 kb of the Leh66b region or 120 kb of the Leh66a region were isolated. Rsk3 covers about 65 kb of the Leh66b region and appears to be incomplete at its 5'-end. A correlation of the physical map provided here with the genetic mapping of Tcr reported previously suggests that Tcr is most likely encoded within a fragment of 30 kb upstream or 20 kb downstream of Rsk3. These data will facilitate the isolation of Tcr, a prerequisite for understanding transmission ratio distortion in mouse.

Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal.

The mouse insulin-like growth factor type 2 receptor (Igf2r) is imprinted and expressed exclusively from the maternally inherited chromosome. To investigate whether methylation could function as the imprinting signal, we have cloned 130 kb from the Igf2r locus and searched for sequences methylated in a parental-specific manner. Two regions have been identified: region 1 contains the start of transcription and is methylated only on the silent paternal chromosome; region 2 is contained in an intron and is methylated only on the expressed maternal chromosome. Methylation of region 1 is acquired after fertilization, in contrast with the methylation of region 2, which is inherited from the female gamete. Methylation of region 2 may mark the maternal Igf2r locus in a manner that could act as an imprinting signal. These data suggest that the expressed locus carries a potential imprinting signal and imply that methylation is necessary for expression of the Igf2r gene.

Conservation of a maternal-specific methylation signal at the human IGF2R locus.

The human IGF2R gene has been reported to be either biallelically or very rarely monoallelically expressed, in contrast to the maternally expressed mouse counterpart. We describe here an analysis of the 5' portion of the human IGF2R gene and show that it contains a maternally methylated CpG island in the second intron. A similar maternally methylated intronic element has been proposed to be the imprinting box for the mouse gene and although the relevance of this element has yet to be directly demonstrated, methylation has been reported to be essential to maintain allele-specific expression of imprinted genes. Allelic expression analysis of human IGF2R in 70 lymphoblastoid cell lines identified only one line showing monoallelic expression. Thus, in this tissue monoparental methylation of the IGF2R gene does not correlate with allele-specific expression. We also confirm here that the human IGF2R gene is located in an asynchronously replicating chromosomal region, as are all other imprinted genes so far analyzed. The mouse and human IGF2R intronic CpG islands both contain numerous large direct repeats that are methylated following maternal, but not paternal, transmittance. Thus features that attract maternal-specific methylation are conserved between the mouse and human genes. Since these intronic CpG islands share organizational rather than sequence homology, this suggests that secondary DNA structure may play a role in attracting a maternal methylation imprint.

Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant.

Chromosomal abnormalities associated with hypomethylation of classical satellite regions are characteristic for the ICF immunodeficiency syndrome. We, as well as others, have found that these effects derive from mutations in the DNMT3B DNA methyltransferase gene. Here we examine further the molecular phenotype of ICF cells and report several examples of extensive hypomethylation that are associated with advanced replication time, nuclease hypersensitivity and a variable escape from silencing for genes on the inactive X and Y chromosomes. Our analysis suggests that all genes on the inactive X chromosome may be extremely hypomethylated at their 5' CpG islands. Our studies of G6PD in one ICF female and SYBL1 in another ICF female provide the first examples of abnormal escape from X chromosome inactivation in untransformed human fibroblasts. XIST RNA localization is normal in these cells, arguing against an independent silencing role for this RNA in somatic cells. SYBL1 silencing is also disrupted on the Y chromosome in ICF male cells. Increased chromatin sensitivity to nuclease was found at all hypomethylated promoters examined, including those of silenced genes. The persistence of inactivation in these latter cases appears to depend critically on delayed replication of DNA because escape from silencing was only seen when replication was advanced to an active X-like pattern.