Emily L. Niemitz

Association of In Vitro Fertilization with Beckwith-Wiedemann Syndrome and Epigenetic Alterations of LIT1 and H19

Recent data in humans and animals suggest that assisted reproductive technology (ART) might affect the epigenetics of early embryogenesis and might cause birth defects. We report the first evidence, to our knowledge, that ART is associated with a human overgrowth syndrome-namely, Beckwith-Wiedemann syndrome (BWS). In a prospective study, the prevalence of ART was 4.6% (3 of 65), versus the background rate of 0.8% in the United States. A total of seven children with BWS were born after ART-five of whom were conceived after intracytoplasmic sperm injection. Molecular studies of six of the children indicate that five of the six have specific epigenetic alterations associated with BWS-four at LIT1 and one at both LIT1 and H19. We discuss the implications of our finding that ART is associated with human overgrowth, similar to the large offspring syndrome reported in ruminants.

BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.

Epigenetics and Assisted Reproductive Technology: A Call for Investigation

A surprising set of recent observations suggests a link between assisted reproductive technology (ART) and epigenetic errors--that is, errors involving information other than DNA sequence that is heritable during cell division. An apparent association with ART was found in registries of children with Beckwith-Wiedemann syndrome, Angelman syndrome, and retinoblastoma. Here, we review the epidemiology and molecular biology behind these studies and those of relevant model systems, and we highlight the need for investigation of two major questions: (1) large-scale case-control studies of ART outcomes, including long-term assessment of the incidence of birth defects and cancer, and (2) investigation of the relationship between epigenetic errors in both offspring and parents, the specific methods of ART used, and the underlying infertility diagnoses. In addition, the components of proprietary commercial media used in ART procedures must be fully and publicly disclosed, so that factors such as methionine content can be assessed, given the relationship in animal studies between methionine exposure and epigenetic changes.

Epigenetic Alterations of H19 and LIT1 Distinguish Patients with Beckwith-Wiedemann Syndrome with Cancer and Birth Defects

Beckwith-Wiedemann syndrome (BWS) is a congenital cancer-predisposition syndrome associated with embryonal cancers, macroglossia, macrosomia, ear pits or ear creases, and midline abdominal-wall defects. The most common constitutional abnormalities in BWS are epigenetic, involving abnormal methylation of either H19 or LIT1, which encode untranslated RNAs on 11p15. We hypothesized that different epigenetic alterations would be associated with specific phenotypes in BWS. To test this hypothesis, we performed a case-cohort study, using the BWS Registry. The cohort consisted of 92 patients with BWS and molecular analysis of both H19 and LIT1, and these patients showed the same frequency of clinical phenotypes as those patients in the Registry from whom biological samples were not available. The frequency of altered DNA methylation of H19 in patients with cancer was significantly higher, 56% (9/16), than the frequency in patients without cancer, 17% (13/76; P=.002), and cancer was not associated with LIT1 alterations. Furthermore, the frequency of altered DNA methylation of LIT1 in patients with midline abdominal-wall defects and macrosomia was significantly higher, 65% (41/63) and 60% (46/77), respectively, than in patients without such defects, 34% (10/29) and 18% (2/11), respectively (P=.012 and P=.02, respectively). Additionally, paternal uniparental disomy (UPD) of 11p15 was associated with hemihypertrophy (P=.003), cancer (P=.03), and hypoglycemia (P=.05). These results define an epigenotype-phenotype relationship in BWS, in which aberrant methylation of H19 and LIT1 and UPD are strongly associated with cancer risk and specific birth defects.

Microdeletion of LIT1 in Familial Beckwith-Wiedemann Syndrome

Beckwith-Wiedemann syndrome (BWS), which causes prenatal overgrowth, midline abdominal wall defects, macroglossia, and embryonal tumors, is a model for understanding the relationship between genomic imprinting, human development, and cancer. The causes are heterogeneous, involving multiple genes on 11p15 and including infrequent mutation of p57(KIP2) or loss of imprinting of either of two imprinted gene domains on 11p15: LIT1, which is near p57(KIP2), or H19/IGF2. Unlike Prader-Willi and Angelman syndromes, no chromosomal deletions have yet been identified. Here we report a microdeletion including the entire LIT1 gene, providing genetic confirmation of the importance of this gene region in BWS. When inherited maternally, the deletion causes BWS with silencing of p57(KIP2), indicating deletion of an element important for the regulation of p57(KIP2) expression. When inherited paternally, there is no phenotype, suggesting that the LIT1 RNA itself is not necessary for normal development in humans.

Quantitative trait loci modulate neutrophil infiltration in the liver during LPS-induced inflammation.

A crucial aspect of the inflammatory response is the recruitment of activated neutrophils (PMN) to the site of damage. Lytic enzymes and oxygen radicals released by PMN are important in clearing an infection or cellular debris, but can also produce host tissue damage. Failure to properly regulate the inflammatory response contributes to a variety of human diseases like sepsis and multiple organ dysfunction syndrome, the leading cause of morbidity and mortality in surgical intensive care units. Many aspects of human disease pathology, including hepatic PMN infiltration, can be recapitulated in mice using an endotoxic shock model. Six quantitative trait loci that predispose to high infiltration of PMN in hepatic sinusoids after high‐dose endotoxin administration were provisionally identified. Two of these loci, Hpil and Hpi2 on mouse chromosomes 5 and 13, were mapped to the significant and highly significant level using a low‐resolution genome scan on 122 intercross animals. These loci interact epistatically to produce a high degree of PMN infiltration. Intercross and recombinant inbred strain mice with a specific genotype at these loci always had a high infiltration response, indicating that genotype analysis at just these two loci can accurately predict a high PMN infiltration response. Genetic predisposition to the degree of PMN infiltration in the inflammatory response in mice suggests that analogous genetic mechanisms occur in human beings that could be used for diagnostic purposes.— Matesic, L. E., Niemitz, E. L., De Maio, A., Reeves, R. H. Quantitative trait loci modulate neutrophil infiltration in the liver during LPS‐induced inflammation. FASEB J. 14, 2247–2254 (2000)

Children with Idiopathic Hemihypertrophy and Beckwith-Wiedemann Syndrome Have Different Constitutional Epigenotypes Associated with Wilms Tumor

Idiopathic hemihypertrophy (IH) is a congenital overgrowth syndrome associated with an increased risk of embryonal cancers in childhood. A related developmental disorder is Beckwith-Wiedemann syndrome (BWS), which increases risk for embryonal cancers, including Wilms tumor. Constitutional epigenetic alterations associated with BWS have been well characterized and include epigenetic alterations of imprinted genes on 11p15. The frequency of hypermethylation of H19 in children with IH and Wilms tumor, 20% (3/15), was significantly lower than the frequency in children with BWS and Wilms tumor, 79% (11/14; P = .0028). These results indicate that children with IH and Wilms tumor have different constitutional epigenotypes from those of children with BWS and Wilms tumor.