No loss of genomic imprinting of IGF-II and H19 in placentas of diabetic pregnancies with fetal macrosomia

Abstract

Objectives

Fetal macrosomia is a common complication of maternal diabetes mellitus and is associated with substantial morbidity, but the precise cellular and molecular mechanisms that induce fetal macrosomia are not well understood. The imprinted genes IGF-II and H19 are crucial for placental development and fetal growth. The term placentas from diabetic pregnancies express more insulin-like growth factor II (IGF-II) than those from normal pregnancies. Deregulation of their imprinting status is observed in the macrosomia-associated syndrome, the Beckwith–Wiedemann syndrome. The aim of this study was to determine whether loss of imprinting hence biallelic expression was also a hallmark of macrosomia in diabetic pregnancies.

Design and methods

IGF-II and H19 maternal and paternal expressions were studied in placentas from two groups of type 1 diabetic mothers: one with macrosomic babies and the other with babies of normal weight. Maternal or paternal allele specific expressions were defined by using DNA polymorphic markers of the IGF-II and H19 genes. RFLP analysis was performed on PCR products from genomic DNA of the father, the mother and the child, and on RT-PCR products from placental mRNA.

Results

RFLP analysis showed that the IGF-II gene remains paternally expressed and the H19 gene remains maternally expressed in all placentas examined, independently of the birth weight status.

Conclusions

These results suggest that, in contrast with Beckwith–Wiedemann syndrome-associated macrosomia, loss of imprinting for IGF-II or H19 is not a common feature of diabetic pregnancies associated with macrosomia.

Introduction

Fetal macrosomia is a common complication of maternal diabetes mellitus and is associated with substantial morbidity, but the precise cellular and molecular mechanisms that induce fetal macrosomia are not well understood. The macrosomia or accelerated fetal growth seen in infants of diabetic mothers is due to a perturbation of a putative placental-fetal growth axis involving growth hormone and insulin-like growth factors. The growth and development of the placenta is critical to fetal growth and development; however, little is known regarding the mechanisms controlling placental growth and development [1]. Despite a good metabolic control the diabetic pregnancy is characterized by a high rate of macrosomia, and a high incidence of malformations and stillbirths [2]. Fetuses developing in diabetic pregnancies receive a large influx of glucose, which in turn may stimulate the expression of insulin-like growth factor II (IGF-II) sequences in placenta, resulting in higher utilization of glucose and overgrowth of placenta [3].

Insulin-like growth factors I and II (IGF-I and IGF-II) are ubiquitous peptides that share structural homology with insulin and have been implicated in processes that control fetal growth. Studies of knockout mice have largely defined the essential roles of the insulin-like growth factors (IGF-I and IGF-II), insulin and their receptors in embryonic and fetal growth, and have provided compelling evidence that increased IGF-II gene expression and/or abundance can stimulate excessive fetal somatic growth [4]. Transgenic mice overexpressing insulin-like growth factor-II in beta cells develop type 2 diabetes [5]. Studies of IGF levels in pregnancies complicated by diabetes and macrosomia have shown conflicting results. By RNA dot blot analysis, the term placentas from gestational diabetic pregnancies were shown to express more insulin-like growth factor II (IGF-II) than those from normal pregnancies [6]. Macrosomia is associated with high levels of plasma maternal IGF-I and –II [7]. Likewise, cord blood IGF-II levels were 2-fold higher in babies of diabetic mothers [8]. However another study showed that cord serum IGF-II levels are elevated in diabetic pregnancies but without a concomitant increase in placental IGF-II levels [9].

Genomic imprinting is an epigenetic phenomenon that results in monoallelic expression of certain genes in a parent-of-origin-dependent manner. It has been suggested to be one of the most important pathways involved in the development and function of the placenta in eutherian mammals. To date, some eighty genes have been found to be controlled by imprinting in humans and mice. One of the most studied imprinted domain on human chromosome 11p15, the IGF-II-H19 domain, encodes insulin-like growth factor 2 (IGF-II) and H19. These imprinted genes which are coordinately regulated and expressed from only one of the parental chromosomes, are crucial for placental development and fetal growth [10]. IGF-II is a 7.5-kD polypeptide known to be involved in the regulation of growth and differentiation, expressed from the paternal allele. H19 is one of the most abundant mRNAs in foetus and placenta, presumably active as RNA, and is believed to function as a tumour suppressor gene, expressed from the maternal allele. A polymorphic functional imprinting has been observed in humans for the IGF-II gene in blood cells. Monoallelic expression of IGF-II is associated with expression of the H19 gene, while biallelic expression of IGF-II is accompanied with loss of H19 expression [11].

The IGF-II gene is usually expressed only by the paternally derived allele however, when this imprinting is erased and IGF-II expression is biallelic, fetal overgrowth ensues. Epigenetic deregulation of imprinting is observed in congenital diseases of aberrant growth such as Beckwith–Wiedemann (BSW) and Silver–Russell syndromes, or transient neonatal diabetes mellitus (TNDM), a congenital disease with intrauterine growth retardation and a transient lack of insulin [12] and in many human cancers [13], [14]. Such increased IGF-II expression would appear to explain the overgrowth in BSW. Indeed, a loss of imprinting (LOI) of IGF-II, with subsequent increased levels of IGF-II mRNA, resulting from biallelic gene expression, was found in BWS [15], an endocrinopathy characterized by fetal and post-natal overgrowth with macrosomia [12].

Whether diabetic macrosomia, like BWS macrosomia, is associated with epigenetic deregulation of imprinting at the IGF-II-H19 cluster has not been studied to date. The aim of this study was to determine whether loss of imprinting (LOI) hence biallelic expression could explain the increased levels of IGF-II [6] in placentas of diabetic mothers according to the presence or absence of fetal macrosomia.